Novel rfrp-3 and dna thereof

ABSTRACT

The RFRP-3 peptide of the present invention which is an agent for promoting prolactin secretion is useful as a prophylactic and/or therapeutic agent for various diseases associated with prolactin secretion, such as hypoovarianism, seminal vesicle hypoplasia, menopausal syndrome and hypothyroidism.

TECHNICAL FIELD

[0001] The present invention relates to a novel polypeptide havingeffect in regulating prolactin secretion (hereinafter, sometimesabbreviated to “RFRP-3”), a polynucleotide encoding the polypeptide, amethod of producing RFRP-3, use of RFRP-3 or a DNA thereof, and so on.

BACKGROUND ART

[0002] A large number of hormones and neurotransmitters are regulatingthe functions of the living body through specific receptors present inthe cell membrane. Many of these receptors perform intracellular signaltransduction through the activation of guanine nucleotide-bindingproteins (hereinafter, sometimes abbreviated to “G proteins”) to whichthey are coupled. Since these receptors have a common structure withseven transmembrane domains, they are called G protein-coupled receptorsor seven times transmembrane type receptors (7TMRs).

[0003] The hypothalamus-pituitary system is one of those passways whichthe above-mentioned hormones/neurotransritters and G protein-coupledreceptors regulate the functions of the living body. The secretion ofpituitary hormones from the pituitary is regulated by hypothalamichormones, and the functions of target cells/organs are regulated throughpituitary hormones released into the blood. The regulation of functionsimportant for the living body, such as the maintenance of homeostasis,development of the reproductive system or individuals, regulation ofmetabolism or growth, and so forth is performed through this passway.

[0004] The secretion of pituitary hormones is regulated by positive ornegative feedback mechanisms involving hypothalamic hormones andperipheral hormones secreted from target internal secretion glands.

[0005] It is known that these hormones, factors and their receptors arenot localized in the hypothalamus-pituitary system but, in general,distributed widely in the brain. Therefore, it is considered thatsubstances called hypothalamic hormones are functioning asneurotransmitters or neuromodulators in the central nervous system.

[0006] These hormones, factors and their receptors are distributedsimilarly in peripheral tissues, and are considered to have importantfunctions individually.

[0007] Under circumstances, development of medicines that regulate thesecretion of pituitary hormones from the pituitary through regulation offunctions of the living body by a G protein-coupled receptor and itsligand, particularly through regulation of hypothalamic hormonesecretion, has been desired.

[0008] A peptide that regulates prolactin release is disclosed in NatureCell Biology, Vol. 2, October 2000, pp. 703-708.

[0009] Secretory peptides designated RFRP-1, RFRP-2 and RFRP-3, and a Gprotein-coupled receptor protein OT7T022 to which these peptides bindare disclosed in WO 00/29441.

[0010] It is disclosed in WO 01/66134 that those secretory peptides havean effect in regulating prolactin secretion.

[0011] It is an object of the present invention to provide a novelpolypeptide which has an excellent effect in regulating release.

DISCLOSURE OF THE INVENTION

[0012] As a result of intensive and extensive researches toward thesolution of the above problem, the present inventors have succeeded inisolating and purifying bovine RFRP-3 which is characterized by havingan RF amide-like structure. The present invention has been achieved uponfurther researches based thereon.

[0013] The present invention provides:

[0014] [1] (1) A peptide consisting of an amino acid sequence which isfrom position 104 (Ala) to position 131 (Phe) of SEQ ID NO: 1 or anamide or ester thereof, or a salt thereof, (2) a peptide consisting ofan amino acid sequence which is from position 101 (Ser) to position 131(Phe) of SEQ ID NO: 1 or an amide or ester thereof, or a salt thereof;or (3) a peptide consisting of an amino acid sequence which is fromposition 104 (Ala) to position 131 (Phe) of SEQ ID NO: 14 or an amide orester thereof, or a salt thereof,

[0015] [2] (1) A peptide consisting of an amino acid sequence which isfrom position 125 (Pro) to position 131 (Phe) of SEQ ID NO: 1 or anamide or ester thereof, or a salt thereof; (2) a peptide consisting ofan amino acid sequence which is from position 126 (Asn) to position 131(Phe) of SEQ ID NO: 1 or an amide or ester thereof, or a salt thereof;or (3) a peptide consisting of an amino acid sequence which is fromposition 127 (Leu) to position 131 (Phe) of SEQ ID NO: 1 or an amide orester thereof, or a salt thereof;

[0016] [3] An amide of the peptide of [1] or [2] above, or a salt of theamide;

[0017] [4] The peptide of [1] or [2] above wherein the C-terminalcarboxyl group is amidated, or a salt thereof;

[0018] [5] A polynucleotide comprising a polynucleotide encoding thepeptide of [1] above;

[0019] [6] A polynucleotide comprising a polynucleotide encoding thepeptide of [2] above;

[0020] [7] The polynucleotide of [5] or [6] above, which is a DNA;

[0021] [8] The polynucleotide of [5] above, which consists of (1) anucleotide sequence which is from position 310 to position 393 of SEQ IDNO: 2, (2) a nucleotide sequence which is from position 301 to position393 of SEQ ID NO: 2, or (3) a nucleotide sequence which is from position310 to position 393 of SEQ ID NO: 15;

[0022] [9] The polynucleotide of [6] above, which consists of (1) anucleotide sequence which is from position 373 to position 393 of SEQ IDNO: 2, (2) a nucleotide sequence which is from position 376 to position393 of SEQ ID NO: 2, or (3) a nucleotide sequence which is from position379 to position 393 of SEQ ID NO: 2;

[0023] [10] A recombinant vector comprising the polynucleotide of [5] or[6] above;

[0024] [11] A transformant transformed with the recombinant vector of[10] above;

[0025] [12] A method for producing the peptide of [1] or [2] above or anamide or ester thereof, or a salt thereof, comprising culturing thetransformant of [11] above and allowing the polypeptide of [1] or [2]above to be produced.

[0026] [13] A medicine comprising the peptide of [1] or [2] above or anamide or ester thereof, or a salt thereof.

[0027] [14] A medicine comprising the polynucleotide of [5] or [6]above;

[0028] [15] The medicine of [13] or [14] above, which is an agent forprolactin secretion;

[0029] [16] The medicine of [13] or [14] above, which is an agent forpromoting prolactin secretion;

[0030] [17] The medicine of [13] or [14] above, which is an inhibitorfor prolactin secretion;

[0031] [18] The medicine of [16] above, which is a prophylactic and/ortherapeutic agent for hypoovarianism, seminal vesicle hypoplasia,osteoporosis, menopausal syndrome, hypogalactia, hypothyroidism or renalinsufficiency;

[0032] [19] The medicine of [17] above, which is a prophylactic and/ortherapeutic agent for hyperprolactinemia, pituitary adenoma,diencephalic tumor, menstrual disorder, stresses, autoimmune diseases,prolactinoma, sterility, impotence, amenorrhea, galactorrhea,acromegaly, Chiari-Frommel syndrome, Argonz-del Castillo syndrome,Forbes-Albright syndrome, breast cancer lymphoma, Sheehan's syndrome orspermatogenic disorder,

[0033] [20] The medicine of [13] or [14] above, which is a promotingagent for milk secretion in a mammal.

[0034] [21] The medicine of [13] or [14] above, which is an agent fortesting the function of prolactin secretion;

[0035] [22] An antibody to the peptide of [1] or [2] above or an amideor ester thereof, or a salt thereof;

[0036] [23] A medicine comprising the antibody of [22] above;

[0037] [24] The medicine of [23] above, which is a prophylactic and/ortherapeutic agent for hyperprolactinemia, pituitary adenoma,diencephalic tumor, menstrual disorder, stresses, autoimmune diseases,prolactinoma, sterility, impotence, amenorrhea, galactorrhea,acromegaly, Chiari-Frommel syndrome, Argonz-del Castillo syndrome,Forbes-Albright syndrome, breast cancer lymphoma or Sheehan's syndromeor spermatogenic disorder,

[0038] [25] A diagnostic agent comprising the antibody of [22] above;

[0039] [26] The diagnostic agent of [25] above, which is a diagnosticagent for hypoovarianism, seminal vesicle hypoplasia, osteoporosis,menopausal syndrome, hypogalactia, hypothyroidism or renalinsufficiency, hyperprolactinemia, pituitary adenoma, diencephalictumor, menstrual disorder, stresses, autoimmune diseases, prolactinoma,sterility, impotence, amenorrhea, galactorthea, acromegaly,Chiari-Frommel syndrome, Argonz-del Castillo syndrome, Forbes-Albrightsyndrome, breast cancer lymphoma or Sheehan's syndrome or spermatogenicdisorder.

[0040] [27] A diagnostic agent comprising the polynucleotide of [5] or[6] above;

[0041] [28] The diagnostic agent of [27] above, which is a diagnosticagent for hypoovarianism, seminal vesicle hypoplasia, osteoporosis,menopausal syndrome, hypogalactia, hypothyroidism or renalinsufficiency, hyperprolactinemia, pituitary adenoma, diencephalictumor, menstrual disorder, stresses, autoimmune diseases, prolactinoma,sterility, impotence, amenorrhea, galactorrhea, acromegaly,Chiari-Frommel syndrome, Argonz-del Castillo syndrome, Forbes-Albrightsyndrome, breast cancer lymphoma or Sheehan's syndrome or spermatogenicdisorder,

[0042] [29] An antisense DNA which comprises a nucleotide sequence, or apart thereof, complementary or substantially complementary to a DNAencoding the peptide of [1] or [2] above, and has an effect capable ofinhibiting the expression of the DNA;

[0043] [30] A medicine comprising the antisense DNA of [29] above;

[0044] [31] The medicine of [30] above, which is a prophylactic and/ortherapeutic agent for hyperprolactinemia, pituitary adenoma,diencephalic tumor, menstrual disorder, stresses, autoimmune diseases,prolactinoma, sterility, impotence, amenorrhea, galactorrhea,acromegaly, Chiari-Frommel syndrome, Argonzdel Castillo syndrome,Forbes-Albright syndrome, breast cancer lymphoma or Sheehan's syndromeor spermatogenic disorder;

[0045] [32] A method of screening for a compound or a salt thereof thatpromotes or inhibits the activity of the peptide of [1] or [2] above oran amide or ester thereof, or a salt thereof, comprising using thepeptide of [1] or [2] above or an anode or ester thereof, or a saltthereof;

[0046] [33] The screening method of [32] above, further comprising usinga protein comprising an amino acid sequence identical or substantiallyidentical to the amino acid sequence represented by SEQ ID NO: 37 or asalt of the protein, or a partial peptide of the protein, or an amide orester of the partial peptide, or a salt of the partial peptide.

[0047] [34] The screening method of [32] above, further comprising usinga protein consisting of the amino acid sequence represented by SEQ IDNO: 37 or SEQ ID NO: 54 or a salt of the protein, or a partial peptideof the protein, or an amide or ester of the partial peptide, or a saltof the partial peptide;

[0048] [35] A kit for screening for a compound or a salt thereof thatpromotes or inhibits the activity of the peptide of [1] or [2] above oran amide or ester thereof, or a salt thereof, comprising the peptide of[1] or [2] above or an amide or ester thereof, or a salt thereof;

[0049] [36] A compound or a salt thereof that promotes or inhibits theactivity of the peptide of [1] or [2] above or an amide or esterthereof, or a salt thereof, wherein the compound or salt thereof isobtainable by the screening method of [32] above or the screening kit of[35] above;

[0050] [37] A medicine comprising a compound or a salt thereof thatpromotes or inhibits the activity of the peptide of [1] or [2] above oran amide or ester thereof, or a salt thereof;

[0051] [38] An agent for promoting prolactin secretion comprising acompound or a salt thereof that promotes the activity of the peptide of[1] or [2] above or an amide or ester thereof, or a salt thereof;

[0052] [39] A prophylactic and/or therapeutic agent for hypoovarianism,seminal vesicle hypoplasia, osteoporosis, menopausal syndrome,hypogalactia, hypothyroidism or renal insufficiency, comprising acompound or a salt thereof that promotes the activity of the peptide of[1] or [2] above or an amide or ester thereof, or a salt thereof;

[0053] [40] A promoting agent for milk secretion in a mammal, comprisinga compound or a salt thereof that promotes the activity of the peptideof [1] or [2] above or an amide or ester thereof, or a salt thereof;

[0054] [41] An inhibitor for prolactin secretion comprising a compoundor a salt thereof that inhibits the activity of the peptide of [1] or[2] above or an amide or ester thereof, or a salt thereof;

[0055] [42] A prophylactic and/or therapeutic agent forhyperprolactinemia, pituitary adenoma, diencephalic tumor, menstrualdisorder, stresses, autoimmune diseases, prolactinoma, sterility,impotence, amenorrhea, galactorrhea, acromegaly, Chiari-Frommelsyndrome, Argonz-del Castillo syndrome, Forbes-Albright syndrome, breastcancer lymphoma, Sheehan's syndrome or spermatogenic disorder.

[0056] [43] A method of promoting prolactin secretion, comprisingadministering to a mammal an effective amount of (i) the peptide of [1]or [2] above or an amide or ester thereof, or a salt thereof, (ii) thepolynucleotide of [5] or [6] above, or (iii) a compound or a saltthereof that promotes the activity of the peptide of [1] or [2] above oran amide or ester thereof, or a salt thereof;

[0057] [44] A method of preventing and/or treating hypoovarianism,seminal vesicle hypoplasia, osteoporosis, menopausal syndrome,hypogalactia, hypothyroidism or renal insufficiency, comprisingadministering to a mammal an effective amount of (i) the peptide of [1]or [2] above or an amide or ester thereof, or a salt thereof, (ii) thepolynucleotide of [5] or [6] above, or (iii) a compound or a saltthereof that promotes the activity of the peptide of [1] or [2] above oran amide or ester thereof, or a salt thereof;

[0058] [45] A method of inhibiting prolactin secretion, comprisingadministering to a mammal an effective amount of (i) the antibody of[22] above, (ii) the antisense DNA of [29] above, or (iii) a compound ora salt thereof that inhibits the activity of the peptide of [1] or [2]above or an amide or ester thereof, or a salt thereof;

[0059] [46] A method of preventing and/or treating hyperprolactinemia,pituitary adenoma, diencephalic tumor, menstrual disorder, stresses,autoimmune diseases, prolactinoma, sterility, impotence, amenorrhea,galactorrhea, acromegaly, Chiari-Frommel syndrome, Argonzdel Castillosyndrome, Forbes-Albright syndrome, breast cancer lymphoma, Sheehan'ssyndrome or spermatogenic disorder, comprising administering to a mammalan effective amount of (i) the antibody of [22] above, (ii) theantisense DNA of [29] above, or (iii) a compound or a salt thereof thatinhibits the activity of the peptide of [1] or [2] above or an amide orester thereof, or a salt thereof;

[0060] [47] Use of (i) the peptide of [1] or [2] above or an amide orester thereof, or a salt thereof, (ii) the polynucleotide of [5] or [6]above, or (iii) a compound or a salt thereof that promotes the activityof the peptide of [1] or [2] above or an amide or ester thereof, or asalt thereof, for manufacturing a prolactin secretion-promoting agent;

[0061] [48] Use of (i) the peptide of [1] or [2] above or an amide orester thereof, or a salt thereof, (ii) the polynucleotide of [5] or [6]above, or (iii) a compound or a salt thereof that promotes the activityof the peptide of [1] or [2] above or an amide or ester thereof, or asalt thereof, for manufacturing a prophylactic and/or therapeutic agentfor hypoovarianism, seminal vesicle hypoplasia, osteoporosis, menopausalsyndrome, hypogalactia, hypothyroidism or renal insufficiency;

[0062] [49] Use of (i) the antibody of [22] above, (ii) the antisenseDNA of [29] above, or (iii) a compound or a salt thereof that inhibitsthe activity of the peptide of [1] or [2] above or an amide or esterthereof, or a salt thereof, for manufacturing an inhibitor of prolactinsecretion; and

[0063] [50] Use of (i) the antibody of [22] above, (ii) the antisenseDNA of [29] above, or (iii) a compound or a salt thereof that inhibitsthe activity of the peptide of [1] or [2] above or an amide or esterthereof, or a salt thereof, for manufacturing a prophylactic and/ortherapeutic agent for hyperprolactinemia, pituitary adenoma,diencephalic tumor, menstrual disorder, stresses, autoimmune diseases,prolactinoma, sterility, impotence, amenorrhea, galactorrhea,acromegaly, Chiari-Frommel syndrome, Argonz-del Castillo syndrome,Forbes-Albright syndrome, breast cancer lymphoma, Sheehan's syndrome orspermatogenic disorder.

BRIEF DESCRIPTION OF THE DRAWINGS

[0064]FIG. 1 shows the nucleotide sequence of a DNA encoding thepolypeptide of the present invention (human type) obtained in ReferenceExample 2, and the amino acid sequence deduced from the nucleotidesequence.

[0065]FIG. 2 is a chart showing the hydrophobic plot of the polypeptideof the present invention.

[0066]FIG. 3 shows the nucleotide sequence of a DNA encoding thepolypeptide of the present invention (human type) obtained in ReferenceExample 3, and the amino acid sequence deduced from the nucleotidesequence.

[0067]FIG. 4 shows the nucleotide sequence of a DNA encoding thepolypeptide of the present invention (bovine type) obtained in ReferenceExample 4, and the amino acid sequence deduced from the nucleotidesequence.

[0068]FIG. 5 shows the nucleotide sequence of a DNA encoding thepolypeptide of the present invention (rat type) obtained in ReferenceExample 5, and the amino acid sequence deduced from the nucleotidesequence.

[0069]FIG. 6 shows comparison of the amino acid sequences of thepolypeptides of the present invention obtained in Reference Examples 3,4 and 5.

[0070]FIG. 7 shows the amino acid sequence of the polypeptide of thepresent invention (mouse type) obtained in Reference Example 6 and thenucleotide sequence of a DNA encoding the polypeptide.

[0071]FIG. 8 shows the figure which shows the reactivity of peptides torOT7T022L receptor-expressing CHO cells examined with a cytosensor inReference Example 7. In this Figure, - represents MPHSFANLPLRF amide(SEQ ID NO: 39) and Δ-Δ represents VPNLPQRF amide (SEQ ID NO: 40).

[0072]FIG. 9 shows the figure which shows cAMP production inhibitoryactivity of MPHSFANLPLRFamide (SEQ ID NO: 39) and VPNLPQRFamide (SEQ IDNO: 40) to rOT7T022L-expressing CHO cells examined in Reference Example10. In this Figure, □-□ represents MPHSFANLPLRFamide (SEQ ID NO: 39) and- represents VPNLPQRF amide (SEQ ID NO: 40).

[0073]FIG. 10 shows the measurement result of the level of the plasmaprolactin determined in Example A1. In this Figure, - represent theprolactin levels of the group to which the peptide represented by SEQ IDNO: 39 was administered in PBS; ◯-◯ represent the prolactin levels ofthe control group to which PBS alone was administered.

[0074] The time point of the administration is taken as 0 min. Mark *represents significance level: p<0.05 and mark ** representssignificance level: p<0.01.

[0075]FIG. 11 shows the result of the reactivity of RF amide-relatedpeptides examined by competitive ELA using anti-rat RFRP-1 monoclonalantibody 1F3 in Reference Example 13.

[0076] To anti-mouse IgGAM antibody-coated 96-well plates, 50 μl ofanti-rat RFRP-1 monoclonal antibody 1F3 and 50 μl of each peptide atconcentrations indicated in the axis of abscissas were added. After16-hr incubation at 4° C., HRP-rat RFRP-1 was added and incubated foranother 2 hr at room temperature. After washing the plates, HRP activitywas determined at an absorbance of 450 nm. “B” shows the absorbance whenthe peptide was added; “B₀” shows the absorbance when the peptide wasnot added.

[0077] In this Figure, -- represents a peptide consisting of an aminoacid sequence which is from position 83 (Val) to position 94 (Phe) ofSEQ ID NO: 50 wherein the C-terminal carboxyl group is amidated(VPHSAANLPLRF-NH₂); -▴- represents a peptide consisting of an amino acidsequence which is from position 90 (Leu) to position 94 (Phe) of SEQ IDNO: 50 wherein the C-terminal carboxyl group is amidated (LPLRF-NH₂);-▪- represents a peptide consisting of an amino acid sequence which isfrom position 124 (Val) to position 131 (Phe) of SEQ ID NO: 1 whereinthe C-terminal carboxyl group is amidated (VPNLPQRF-NH₂); and -□-represents a peptide consisting of an amino acid sequence which is fromposition 128 (Pro) to position 131 (Phe) of SEQ ID NO: 1 wherein theC-terminal carboxyl group is amidated (PQRF-NH₂).

[0078]FIG. 12 shows the chromatographic pattern of finally purifiedendogenous RFRP-1 from bovine hypothalamus obtained in Example A2. It isshown that the chromatogram of μ RPC C2/C18 SC 2.1/10 at the finalpurification stage. The axis of ordinates represents absorbance and theconcentration of eluted acetonitrile; the axis of abscissas representsretention time. The black columns in the Figure show the RFRP-1-likeimmune activities of individual fractions measured by competitive EIAusing anti-rat RFRP-1 monoclonal antibody 1F3.

[0079]FIG. 13 shows the construction of plasmid pTFCRFRP-1 obtained inExample A4.

[0080]FIG. 14 shows the figure of the inhibitory activities ofindividual peptides against the increase of intracellular cAMP withforskolin treatment examined in Example A8. In this Figure, -◯-represents hRFRP-1-12 (a peptide comprising an amino acid sequence whichis from position 81 (Met) to position 92 (Phe) of SEQ ID NO: 1); -▪-represents hRFRP-1-37 (a peptide comprising an amino acid sequence whichis from position 56 (Ser) to position 92 (Phe) of SEQ ID NO: 1); -▴-represents rRFRP-1-37 (a peptide comprising an amino acid sequence whichis from position 58 (Ser) to position 94 (Phe) of SEQ ID NO: 50); -▴-represents hRFRP-2-12 (a peptide comprising an amino acid sequence whichis from position 101 (Phe) to position 112 (Ser) of SEQ ID NO: 1); -□-represents hRFRP-3-8 (a peptide comprising an amino acid sequence whichis from position 124 (Val) to position 131 (Phe) of SEQ ID NO: 1); -♦-represents PQRFamide (a peptide represented by Pro-Gln-Arg-Phe-NH₂); --represents LPLRFamide (a peptide represented byLeu-Pro-Leu-Arg-Phe-NH₂); and -▴- represents NPFF (a peptide representedby Asn-Pro-Phe-Phe).

[0081]FIG. 15 shows the figure of the effect of pertussis toxin uponactivation of human OT7T022 receptor by RFRP peptides examined inExample A9; the above effect is shown using cAMP production inhibitoryeffect as an indicator.

[0082]FIG. 16 shows the reactivity of RF amide-related peptides incompetitive EIA using anti-rat RFRP-3 monoclonal antibody 7F6. “B” showsthe absorbance when the peptide was added; “B₀” shows the absorbancewhen the peptide was not added.

[0083]FIG. 17 shows the chromatographic pattern of finally purifiedendogenous RFRP-3 from bovine hypothalamus. This chromatographic chartshows the absorbance at 215 nm and the concentration of elutedacetonitrile. The black columns in the Figure show the RFRP-3-likeimmune activities of individual fractions measured by competitive EIAusing anti-rat RFRP-3 monoclonal antibody 7F6.

[0084]FIG. 18 shows the results of N-terminal amino acid analysis forthe finally purified endogenous RFRP-3 sample from bovine hypothalamus.

[0085]FIG. 19 shows the results of determination of the molecular weightof the finally purified endogenous RFRP-3 sample from bovinehypothalamus.

[0086]FIG. 20 shows the MS/MS spectrum of the finally purifiedendogenous RFRP-3 sample purified from bovine hypothalamus measuredusing a pentavalent molecule-related ion (m/z 661) as a precursor ion.

[0087]FIG. 21 shows the reactivity of RF amide-related peptides incompetitive EIA using anti-rat RFRP-3 polyclonal antibody. “B” shows theabsorbance when the peptide was added; “B₀” shows the absorbance whenthe peptide was not added.

BEST MODES FOR CARRYING OUT THE INVENTION

[0088] A polypeptide comprising an amino acid sequence identical orsubstantially identical to the amino acid sequence represented by SEQ IDNO: 1 (hereinafter, sometimes referred to as the “polypeptide of theinvention”) may be a polypeptide derived from cells of any kind (e.g.retinal cells, hepatocytes, splenocytes, nerve cells, glia cells,pancreatic β cells, bone marrow cells, mesangial cells, Langerhan'scells, epidermal cells, epithelial cells, endothelial cells,fibroblasts, fibrous cells, muscle cells, fat cells, immune cells (e.g.macrophages, T cells, B cells, natural killer cells, mast cells,neutrophils, basophils, eosinophils, monocytes), megakaryocytes,synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts,mammary cells, hepatocytes or interstitial cells, or progenitor cells,stem cells or cancer cells of these cells, and so forth) of human orother warm-blooded animals (e.g. guinea pig, rat, mouse, chicken,rabbit, pig, sheep, bovine, monkey, and so forth) or any tissue in whichsuch cells are present, such as brain, various parts of brain (e.g.retina, olfactory bulb, amygdaloid nucleus, cerebral basal nucleus,hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata,cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney,liver, gonad, thyroid, gall-bladder, bone marrow, adrenal gland, skin,muscle, lung, gastrointestinal tracts (e.g. large intestine, smallintestine), blood vessels, heart, thymus, spleen, submandibular gland,peripheral blood, prostate, testis, ovary, placenta, uterus, bone,joint, skeletal muscle, and so forth or hemocyte lineage cells orcultured cells thereof (e.g. MEL, M1, CTLL-2, HT-2, WEH1-3, HL-60,JOSK-1, K562, ML-1, MOLT-3, MOLTA, MOLT-10, CCRF-CEM, TALL-1, Jurkat,CCRT-HSB-2, KE-37, SKW-3, HUT-78, HUT-102, H19, U937, THP-1, BEL, JK-1,CMK, KO-812, MEG-01, and so forth). The polypeptide may also be asynthetic polypeptide.

[0089] Examples of amino acid sequences substantially identical to theamino acid sequence represented by SEQ ID NO: 1 include amino acidsequences having about 70% or more, preferably about 80% or more, morepreferably about 90% or more, still more preferably about 95% or morehomology to the amino acid sequence represented by SEQ ID NO: 1.

[0090] As an amino acid sequence substantially identical to the aminoacid sequence represented by SEQ ID NO: 1, an amino acid sequencecomprising the amino acid sequence which is from position 22 to position180 of the amino acid sequence represented by SEQ ID NO: 1 may be given,for example.

[0091] More specific examples of amino acid sequences substantiallyidentical to the amino acid sequence represented by SEQ ID NO: 1 includethe amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 14, SEQID NO: 18, SEQ ID NO: 33 or SEQ ID NO: 50.

[0092] Examples of polypeptides comprising an amino acid sequencesubstantially identical to the amino acid sequence represented by SEQ IDNO: 1 include those polypeptides which comprise the above-describedamino acid sequence substantially identical to the amino acid sequencerepresented by SEQ ID NO: 1 (e.g. the amino acid sequence represented bySEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 33 or SEQ ID NO:50) and has such an activity of regulating prolactin secretion aspossessed by a polypeptide comprising the amino acid sequencerepresented by SEQ ID NO: 1.

[0093] The term “substantially identical” means that the activities ofpolypeptides are naturally same (e.g. physiologically orpharmacologically). Therefore, it is preferable that the activities ofregulating prolactin secretion should be equivalent (e.g. about 0.1- to100-fold, preferably about 0.5- to 10-fold, more preferably 0.5- to2-fold). However, quantitative factors, such as the degree ofactivities, the molecular weights of polypeptides, may be optionallydifferent.

[0094] The determination of activity of regulating prolactin secretionmay be carried out by known methods. For example, this activity may bemeasured according to the procedures described later in Example 1.

[0095] The polypeptide of the present invention include the so-calledmuteins, such as polypeptides comprising (i) the amino acid sequence ofSEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 33or SEQ ID NO: 50 wherein 1-20 amino acids (preferably 1-15, morepreferably 1-5, and still more preferably 1-3 amino acids) are deletedtherefrom; (ii) the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 8,SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 33 or SEQ ID NO: 50 wherein1-20 amino acids (preferably 1-15, more preferably 1-5, and still morepreferably 1-3 amino acids) are added thereto; (iii) the amino acidsequence of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 18,SEQ ID NO: 33 or SEQ ID NO: 50 wherein 1-20 amino acids (preferably1-15, more preferably 1-5, and still more preferably 1-3 amino acids)are inserted thereinto; (iv) the amino acid sequence of SEQ ID NO: 1,SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 33 or SEQ ID NO:50 wherein 1-20 amino acids (preferably 1-15, more preferably 1-5, andstill more preferably 1-3 amino acids); or (v) an amino acid sequencewhich is a combination of these sequences.

[0096] If the amino acid sequence has such an insertion, deletion orsubstitution as described above, the site of the insertion, deletion orsubstitution is not particularly restricted.

[0097] Specific examples of polypeptides comprising an amino acidsequence substantially identical to the amino acid sequence representedby SEQ ID NO: 2 include a polypeptide comprising the amino acid sequencerepresented by SEQ ID NO: 8, a polypeptide comprising the amino acidsequence represented by SEQ ID NO: 14, a polypeptide comprising theamino acid sequence represented by SEQ ID NO: 18, a polypeptidecomprising the amino acid sequence represented by SEQ ID NO: 33, and apolypeptide comprising the amino acid sequence represented by SEQ ID NO:50.

[0098] The polypeptides in the present specification are expressedaccording to the conventional description of peptides, that is, theN-terminus (amino terminus) at the left end and the C-terminus (carboxylterminus) at the right end. The C-terminus of the polypeptide of theinvention (such as a polypeptide comprising the amino acid sequencerepresented by SEQ ID NO: 4) may be either a carboxyl group (—COOH), acarboxylate (—COO⁻), an amide (—CONH₂) or an ester (—COOR).

[0099] Examples of R of the above ester group include C₁₋₆ alkyl groups(e.g. methyl, ethyl, n-propyl, isopropyl or n-butyl), C₃₋₈ cycloalkylgroups (e.g. cyclopentyl or cyclohexyl), C₆₋₂ aryl groups (e.g. phenylor α-naphthyl), C₇₋₁₄ aralkyl groups such as phenyl-C₁₋₂ alkyl groups(e.g. benzyl or phenethyl) and α-naphthyl-C₁₋₂ alkyl groups (e.g.α-naphthylmethyl). In addition, the ester group also includespivaloyloxymethyl esters that are universally used as oral esters.

[0100] When the polypeptide of the present invention has a carboxylgroup (or carboxylate) at any position other than its C-terminus, thecarboxyl group may be amidated or esterified; such a polypeptide is alsoincluded in the polypeptide of the invention. The ester in this case maybe, for example, any of the esters mentioned above for the C-terminalester.

[0101] Furthermore, the polypeptide of the present invention includesthose polypeptides in which the N-terminal amino acid residue (e.g. Met)is protected by a protective group (e.g. C₁₋₆ acyl group such as C₁₋₆alkanoyl group (e.g. formyl group or acetyl group)); those polypeptidesin which the N-terminal Glu generated through in vivo cleavage ispyroglutaminated; those polypeptides in which a substituent on a sidechain of an amino acid (e.g. —OH, —SH, amino group, imidazole group,indole group, or guannidino group) is protected by an appropriateprotective group (e.g. C₁₋₆ acyl group such as Con alkanoyl group (e.g.formyl group or acetyl group)); and conjugated proteins such as theso-called glycoproteins to which sugar chains are linked. Hereinafter,these polypeptides may sometimes be referred to as the polypeptide ofthe present invention.

[0102] Specific examples of the polypeptide of the present inventioninclude a human-derived polypeptide comprising the amino acid sequencerepresented by SEQ ID NO: 1, a human-derived polypeptide comprising theamino acid sequence represented by SEQ ID NO: 8, a bovine-derivedpolypeptide comprising the amino acid sequence represented by SEQ ID NO:14, a rat-derived polypeptide comprising the amino acid sequencerepresented by SEQ ID NO: 18, a mouse-derived polypeptide comprising theamino acid sequence represented by SEQ ID NO: 33, and a rat-derivedpolypeptide comprising the amino acid sequence represented by SEQ ID NO:50. For example, a human-derived polypeptide comprising the amino acidsequence represented by SEQ ID NO: 1, a human-derived polypeptidecomprising the amino acid sequence represented by SEQ ID NO: 8, and abovine-derived polypeptide comprising the amino acid sequencerepresented by SEQ ID NO: 14 are used preferably.

[0103] The RFRP-3 of the present invention consists of (i) an amino acidsequence which is from position 104 (Ala) to position 131 (Phe) of SEQID NO: 1 (SEQ ID NO: 63); (i) an amino acid sequence which is fromposition 101 (Ser) to position 131 (Phe) of SEQ ID NO: 1 (SEQ ID NO:65); or (iii) the amino acid sequence which is from position 104 (Ser)to position 131 (Phe) of SEQ ID NO: 14 (SEQ ID NO: 67).

[0104] The RFRP-3 of the present invention may comprise theabove-described amino acid sequence where 1-5 amino acids (preferably1-3 amino acids) are deleted, or 1-5 amino acids (preferably 1-3 aminoacids) are added, or 1-5 amino acids (preferably 1-3 amino acids) areinserted, or 1-5 amino acids (preferably 1-3 amino acids) aresubstituted with other amino acids; or may comprise an amino acidsequence which is a combination of these amino acid sequences.

[0105] Further, as the RFRP-3 of the present invention, a peptide mayalso be used which comprises at least an amino acid sequence which isfrom position 127 (Leu) to position 131 (Phe) of the amino acid sequencerepresented by SEQ ID NO: 1 or SEQ ID NO: 12 (SEQ ID NO: 69) on itsC-terminal side. Specifically, a peptide consisting of an amino acidsequence which is from position 124 (Val) to position 131 (Phe) of theamino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 12 (SEQ IDNO: 72) (RFRP-3 (8)); a peptide consisting of an amino acid sequencewhich is from position 125 (Pro) to position 131 (Phe) (SEQ ID NO: 71)(RFRP-3 (7)); a peptide consisting of an amino acid sequence which isfrom position 126 (Asn) to position 131 (Phe) of the amino acid sequencerepresented by SEQ BD NO: 1 or SEQ D NO: 12 (SEQ ID NO: 70) (RFRP-3(6)); or a peptide consisting of an amino acid sequence which is fromposition 127 (Leu) to position 131 (Phe) of the amino acid sequencerepresented by SEQ ID NO: 1 or SEQ ID NO: 12 (SEQ ID NO: 69) (RFRP-3(5)) may be used. Among all, the peptide consisting of an amino acidsequence which is from position 125 (Pro) to position 131 (Phe) of theamino acid sequence represented by SEQ ID NO: 1 (SEQ ID NO: 71) (RFRP-3(7)); the peptide consisting of an amino acid sequence which is fromposition 126 (Asn) to position 131 (Phe) of the amino acid sequencerepresented by SEQ ID NO: 1 (SEQ ID NO: 70) (RFRP-3 (6)); or the peptideconsisting of an amino acid sequence which is from position 127 (Leu) toposition 131 (Phe) of the amino acid sequence represented by SEQ ID NO:1 (SEQ ID NO: 69) (RFRP-3 (5)) are used preferably. In particular, thepeptide RFRP-3 (7) consisting of an amino acid sequence which is fromposition 125 (Pro) to position 131 (Phe) of the amino acid sequencerepresented by SEQ ID NO: 1 (SEQ ID NO: 71) is used preferably.Preferably, the C-terminus of these peptides is an amide.

[0106] The C-terminus of the RFRP-3 of the present invention may beeither a carboxyl group (—COOH), a carboxylate (—COO⁻), an amide(—CONH₂) or an ester (—COOR) (where R has the same definition asdescribed above). Among all, those which have an amide (CONH₂) on theirC-terminus are preferable.

[0107] When the RFRP-3 of the present invention has a carboxyl group (orcarboxylate) at any position other than its C-terminus, the carboxylgroup may be amidated or esterified; such RFRP-3 is also included in thepolypeptide of the present invention. The ester in this case may be, forexample, any of the esters mentioned above for the C-terminal ester.

[0108] Further, like the polypeptide of the present invention describedabove, the RFRP-3 of the present invention also includes those peptidesin which the N-terminal amino acid residue (e.g. Met) is protected by aprotective group; those peptides in which the N-terminal Glu generatedthrough in vivo cleavage is pyroglutaminated; those peptides in which asubstituent on a side chain of an amino acid is protected by anappropriate protective group; and conjugated peptides such as theso-called glycopeptides to which sugar chains are linked.

[0109] The RFRP-3 of the present invention has an RF amide structure.The term “RF amide structure” means that the C-terminal of a peptide isarginine-phenylalanine-NH₂.

[0110] As salts of the polypeptide or the RFRP-3 of the presentinvention, salts formed with physiologically acceptable acids (e.g.organic or inorganic acids) or bases (e.g. alkali metals) are used.Especially preferable are physiologically acceptable acid additionsalts. Examples of such salts include salts formed with inorganic acids(e.g. hydrochloric acid, phosphoric acid, hydrobromic acid or sulfuricacid) and salts formed with organic acids (e.g. acetic acid, formicacid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaricacid, citric acid, malic acid, oxalic acid, benzoic acid,methanesulfonic acid or benzenesulfonic acid).

[0111] The polypeptide of the present invention or salts thereof or theRFRP of the invention and salts thereof can be produced from theafore-mentioned cells or tissues of human or other warm-blooded animalsby known purification methods for polypeptides. Alternatively, they canalso be produced by culturing a transformant comprising a DNA describedlater encoding the polynucleotide. They can also be produced inaccordance with the procedures for peptide synthesis which are describedlater.

[0112] In producing the polypeptide or salts thereof or the RFRP orsalts thereof from tissues or cells of human or other mammals, therelevant tissue or cell is homogenized and then the desired polypeptide,and so forth is extracted with acids, and so forth. The desiredpolypeptide, and so forth can be purified and isolated from theresultant extract by a combination of chromatography, such as reversedphase chromatography, ion exchange chromatography and so on.

[0113] It is useful for the synthesis of the polypeptide of the presentinvention or salts thereof or the RFRP of the present invention or saltsthereof that any of the commercial resins available for polypeptidesynthesis. Examples of such resins include chloromethyl resin,hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin,4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAMresin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamideresin, 4-(2′,4′-dimethoxyphenylhydroxymethyl) phenoxy resin, and4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl) phenoxy resin. Using such aresin, amino acids of which their α-amino groups and side chainfunctional groups are protected are condensed on the resin according tothe amino acid sequence of the desired polypeptide by conventionalcondensation methods. At the final stage of the reaction, all protectivegroups are removed simultaneously with the cleavage of the polypeptidefrom the resin. Then, in a highly diluted solution, intramoleculardisulfide bond formation reaction is carried out to obtain thepolypeptide of the invention or a salt thereof of the RFRP of theinvention of a salt thereof.

[0114] With respect to the condensation of the above-described protectedamino acids, it may be utilized that various activators useful forpolypeptide synthesis. Among all, carbodiimide reagents are especiallypreferred. Examples of carbodiimide reagents include DCC,N,N′-diisopropylcarbodiimide, andN-ethyl-N′-(3-dimethylaminoprolyl)carbodiimide. For activation by thesereagents, protected amino acids and a recemization inhibitor (e.g. HOBtor HOOBt) may be directly added to the resin, or protected amino acidsmay be activated in advance in the form of symmetric acid anhydride,HOBt ester or HOOBt ester and then added to the resin.

[0115] The solvent used for the above-mentioned activation of protectedamino acids or the condensation thereof with a resin may beappropriately selected from those solvents known to be useful forpolypeptide (protein) condensation reactions. Examples of usefulsolvents include acid amides (e.g. N,N-dimethylformamide,N,N-dimethylacetamide or N-methylpyrrolidone), halogenated hydrocarbons(e.g. methylene chloride, or chloroform), alcohols (e.g.trifluoroethanol), sulfoxides (e.g. dimethyl sulfoxide), ethers (e.g.pyridine, dioxane, tetrahydrofuran), nitriles (e.g. acetonitrile orpropionitrile), esters (e.g. methyl acetate or ethyl acetate), andsuitable mixtures of these solvents. The reaction temperature may beappropriately selected from the range known to be useful for polypeptidebond-forming reactions; usually, the temperature is selected from therange from about −20° C. to about 50° C. The activated amino acidderivative is usually used in 1.5- to 4-fold excess. If the condensationis found insufficient as a result of test using the ninhydrin reaction,sufficient condensation can be achieved by repeating reactions withoutremoving protective groups. If sufficient condensation cannot beachieved even by repeating reactions, unreacted amino acids may beacetylated with acetic anhydride or acetylimidazole so that they do notaffect subsequent reactions.

[0116] Examples of useful protective groups for the amino group of rawmaterials include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl,4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl,trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl,diphenylphosphinothioyl, and Fmoc.

[0117] The carboxyl group can be protected, for example, in the form ofan alkyl ester (e.g. straight-chain, branched, or cyclic alkyl esterssuch as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, 2-adamantyl, and so on), aralkyl ester (e.g.benzyl, 4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, benzhydryl, andso on), phenacyl ester, benzyloxycarbonylhydrazide,t-butoxycarbonylhydrazide or tritylhydrazide.

[0118] The hydroxyl group of serine can be protected, for example, byesterification or etherification. Examples of suitable groups for thisesterification include lower (C₁₋₆) alkanoyl groups such as acetyl,aroyl groups such as benzoyl, and carbonic acid-derived groups such asbenzyloxycarbonyl and ethyloxycarbonyl. Examples of groups suitable forthe etherification include benzyl, tetaahydropyranyl and t-butyl.

[0119] Examples of protective groups for the phenolic hydroxyl group oftyrosine include Bzl, C₁₂-Bzl, 2-nitrobenzyl, BrZ, and t-butyl.

[0120] Examples of protective groups for the imidazole ring of histidineinclude Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP,benzyloxymethyl, Burn, Boc, Trt and Fmoc.

[0121] Examples of materials with activated carboxyl groups include thecorresponding acid anhydrides, azides and active esters (esters ofalcohols such as pentachchlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenoli cyanomethyl alcohol, p-nitrophenol, HONB,N-hydroxysuccinimide, N-hydroxyphthalimide and HOBt). Examples of rawmaterials with activated amino groups include the correspondingphosphoric acid amides.

[0122] Methods for removing (eliminating) protective groups include, forexample, catalytic reduction in a hydrogen stream in the presence of acatalyst such as Pd black or Pd-carbon, acid treatment with anhydroushydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid,trifluoroacetic acid or mixtures thereof, treatment with a base such asdiiso-propylethylamine, triethylamine, piperidine, piperazine or thelike, and reduction with sodium in liquid ammonia. The eliminationreaction by the above-mentioned acid treatment is generally conducted attemperatures of about −20° C. to about 40° C. In the acid treatment, itis effective to add a cation scavenger such as anisole, phenol,thioanisole, m-cresol, p-cresol, dimethylsulfide, 1,4-butanedithiol or1,2-ethanedithiol. The 2,4-dinitrophenyl group used as the protectivegroup for the imidazole ring of histidine is removed by thiophenoltreatment. The formyl group used as the protective group for the indolering of tryptophan may be removed by the above-mentioned deprotection bythe acid treatment in the presence of 1,2-ethanedithiol,1,4-butanedithiol or the like, or by alkali treatment using dilutesodium hydroxide, dilute ammonia or the like.

[0123] The protection of functional groups in materials that should notbe involved in the reaction, protective groups therefor, the removal ofthese protective groups and the activation of functional groups involvedin the reaction can be appropriately selected from groups or methodsknown in the art.

[0124] An alternative method for obtaining amides of the polypeptide orthe RFRP of the invention comprises, for example, protecting theα-carboxyl group of the C-terminal amino acid by amidation, extendingthe peptide (polypeptide) chain to a desired length on the side of theamino group, preparing a polypeptide with its N-terminal α-amino groupselectively deprotected, preparing a polypeptide with its C-terminalcarboxyl group selectively deprotected, and condensing these twopolypeptides in a mixed solvent such as described above. Details of thiscondensation reaction are the same as described above. Afterpurification of the protected polypeptide thus obtained by condensation,all the protective groups are removed by the method described above tothereby to provide a desired crude polypeptide. This crude polypeptideis purified by various known purification techniques and lyophilized toprovide the desired polypeptide or RFRP in an amide form.

[0125] As a method for obtaining esters of the polypeptide or the RFRPof the invention, for example, the α-caboxyl group of the C-terminalamino acid is condensed with a desired alcohol to prepare thecorresponding amino acid ester, and then this ester is subjected to thesame procedures as described above in the preparation of amides tothereby obtain the desired polypeptide or RFRP in an ester form.

[0126] The RFRP of the invention or salts thereof can be produced byknown methods for peptide synthesis. Alternatively, the RFRP or saltsthereof may be produced by digesting the polypeptide of the presentinvention with an appropriate peptidase. The method for peptidesynthesis may be solid-phase synthesis or liquid-phase synthesis.Briefly, a desired peptide can be produced by condensing a partialpeptide or amino acids capable of constituting the RFRP of the presentinvention with the remaining part thereof and, if the product hasprotective groups, removing the protective groups. Examples ofcondensation methods and methods for removal of protective groups knownin the art include those described in the following references (i) to(v).

[0127] (i) M. Bodanszky & M. A. Ondetti, Peptide Synthesis, IntersciencePublishers, New York, 1966

[0128] (ii) Schroeder & Luebke, The Peptide, Academic Press, New York,1965

[0129] (iii) Nobuo Izumiya et al., Fundamentals and Experiments inPeptide Synthesis, Maruzen, 1975

[0130] (iv) Haruaki Yajima and Shumpei Sakakibara, BiochemicalExperiment Series 1, Polypeptide Chemistry IV, 205, 1977, and

[0131] (v) Haruaki Yajima (ed.), Development of Mediciens (Continued),Vol. 14, Peptide Synthesis, Hirokawa Shoten

[0132] After the reaction, the RFRP of the present invention can beisolated and purified by a combination of conventional purificationtechniques such as solvent extraction, distillation, columnchromatography, liquid chromatography, and recrystallization. If theobtained RFRP is free, it can be converted to a suitable salt by knownmethods or methods based thereon. On the contrary, if the RFRP isobtained in a salt form, it can be converted to be free or another saltby known methods or methods based thereon.

[0133] The polynucleotide encoding the polypeptide of the presentinvention may be any polynucleotide as long as it comprises a nucleotidesequence encoding the polypeptide of the invention (DNA or RNA;preferably, DNA). The polynucleotide may be a DNA or RNA (such as mRNA)encoding the polypeptide of the present invention, and may bedouble-stranded or single-stranded. If the polynucleotide isdouble-stranded, it may be a double-stranded DNA, a double-stranded RNA,or a DNA:RNA hybrid. If the polynucleotide is single-stranded, it may bea sense strand (i.e. coding strand) or an anti-sense strand (i.e.non-coding strand).

[0134] With the polynucleotide encoding the polypeptide of the presentinvention, it is possible to quantitatively determine the mRNA of thepolypeptide of the present invention by such methods as described in NewPCR and Its Application (Extra Issue of Experimental Medicine), 15 (7),1997 or modifications thereof.

[0135] The DNA encoding the polypeptide of the present invention may beany DNA as long as it comprises the above-described nucleotide sequenceencoding the polypeptide of the present invention. The DNA may begenomic DNA, genomic DNA library, cDNA derived from the above-mentionedcells or tissues, cDNA library derived from the above-mentioned cells ortissues, or synthetic DNA.

[0136] Vectors used for library construction may be any vectors such asbacteriophage, plasmid, cosmid, phagemid, and so on. Alternatively,total RNA or mRNA fraction may be prepared from the above-mentionedcells or tissues, followed by direct amplification by reversetranscriptase polymerase chain reaction (hereinafter, abbreviated to“RT-PCR”).

[0137] Specific examples of the DNA encoding the polypeptide of thepresent invention include any DNA comprising the nucleotide sequencerepresented by SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19,SEQ ID NO: 34 or SEQ ID NO: 51; or any DNA which comprises a nucleotidesequence hybridizing to the nucleotide sequence represented by SEQ IDNO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 34 or SEQID NO: 51 under highly stringent conditions and encodes a polypeptidewhich substantially has an activity (e.g. cell stimulatory activity)naturally same to the activity of the polypeptide of the presentinvention.

[0138] As DNAs capable of hybridizing to the nucleotide sequencerepresented by SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19,SEQ ID NO: 34 or SEQ ID NO: 51 under highly stringent conditions, DNAscomprising a nucleotide sequence having about 70% or more, preferablyabout 80% or more, more preferably about 90% or more, still morepreferably about 95% or more homology to the nucleotide sequencerepresented by SEQ ID NO: 2 may be used, for example.

[0139] Hybridization can be carried out according to known methods ormethods based thereon, e.g. those methods described in “MolecularCloning,” 2nd Ed. (J. Sambrook et al., Cold Spring Harbor Lab. Press,1989). When commercial libraries are used, hybridization can be carriedout in accordance with the methods described in the instructionsattached thereto; more preferably, hybridization is carried out underhighly stringent conditions.

[0140] “Highly stringent conditions” refers to, for example, conditionswhere the sodium concentration is about 1940 mM, preferably about 19-20mM, and the temperature is about 50-70° C., preferably about 60-65° C.In particular, conditions where the sodium concentration is about 19 mMand the temperature is about 65° C. are most preferable.

[0141] More specifically, as a DNA encoding a polypeptide comprising theamino acid sequence represented by SEQ ID NO: 1, a DNA comprising thenucleotide sequence represented by SEQ ID NO: 2 may be used, forexample. As a DNA encoding a polypeptide comprising the amino acidsequence represented by SEQ ID NO: 8, a DNA comprising the nucleotidesequence represented by SEQ ID NO: 9 may be used, for example. As a DNAencoding a polypeptide comprising the amino acid sequence represented bySEQ ID NO: 14, a DNA comprising the nucleotide sequence represented bySEQ ID NO: 15 may be used, for example. As a DNA encoding a polypeptidecomprising the amino acid sequence represented by SEQ ID NO: 18, a DNAcomprising the nucleotide sequence represented by SEQ ID NO: 19 may beused, for example. As a DNA encoding a polypeptide comprising the aminoacid sequence represented by SEQ ID NO: 33, a DNA comprising thenucleotide sequence represented by SEQ ID NO: 34 may be used, forexample. As a DNA encoding a polypeptide comprising the amino acidsequence represented by SEQ ID NO: 50, a DNA comprising the nucleotidesequence represented by SEQ ID NO: 51 may be used, for example.

[0142] The polynucleotide encoding the RFRP-3 of the present inventionmay be any polynucleotide as long as it comprises a nucleotide sequenceencoding the RFRP-3 of the invention (DNA or RNA; preferably, DNA). Thepolynucleotide may be a DNA or RNA (such as mRNA) encoding the RFRP-3 ofthe present invention, and may be double-stranded or single-stranded.When the polynucleotide is double-stranded, it may be a double-strandedDNA, a double-stranded RNA, or a DNA:RNA hybrid. When the polynucleotideis single-stranded, it may be a sense strand (i.e. coding strand) or ananti-sense strand (i.e. non-coding strand).

[0143] With the polynucleotide encoding the RFRP-3 of the presentinvention, it is possible to quantitatively determine the mRNA of theRFRP-3 of the present invention by such methods as described in New PCRand Its Application (Extra Issue of Experimental Medicine), 15 (7), 1997or modifications thereof.

[0144] The DNA encoding the RFRP-3 of the present invention may be anyDNA as long as it comprises the above-described nucleotide sequenceencoding the RFRP-3 of the present invention. The DNA may be genomicDNA, genomic DNA library, cDNA derived from the above-mentioned cells ortissues, cDNA library derived from the above-mentioned cells or tissues,or synthetic DNA.

[0145] Specific examples of the DNA encoding the RFRP-3 of the presentinvention include:

[0146] (i) DNAs encoding a peptide consisting of an amino acid sequencewhich is from position 104 (Ala) to position 131 (Phe) of SEQ ID NO: 1(SEQ ID NO: 63); or DNAs comprising a nucleotide sequence hybridizing tothese DNAs under highly stringent conditions;

[0147] (ii) DNAs encoding a peptide consisting of an amino acid sequencewhich is from position 101 (Ser) to position 131 (Phe) of SEQ ID NO: 1(SEQ ID NO: 65); or DNAs comprising a nucleotide sequence hybridizing tothese DNAs under highly stringent conditions;

[0148] (iii) DNAs encoding a peptide consisting of an amino acidsequence which is from position 104 (Ala) to position 131 (Phe) of SEQID NO: 14 (SEQ ID NO: 67); or DNAs comprising a nucleotide sequencehybridizing to these DNAs under highly stringent conditions.

[0149] “Highly stringent conditions” refers to, for example, conditionswherein sodium concentration is about 19-40 mM, preferably about 19-20mM, and temperature is about 50-70° C., preferably about 60-65° C. Inparticular, conditions wherein sodium concentration is about 19 mM andtemperature is about 65° C. are most preferable.

[0150] More specifically;

[0151] (i) as the DNA encoding a peptide consisting of an amino acidsequence which is from position 104 (Ala) to position 131 (Phe) of SEQID NO: 1 (SEQ ID NO: 63), a DNA consisting of a nucleotide sequencewhich is from position 310 to position 393 of the nucleotide sequencerepresented by SEQ ID NO: 2 (SEQ ID NO: 64) may be given, for example;

[0152] (ii) as the DNA encoding a peptide consisting of an amino acidsequence which is from position 101 (Ser) to position 131 (Phe) of SEQID NO: 1 (SEQ ID NO: 65), a DNA consisting of a nucleotide sequencewhich is from position 301 to position 393 of the nucleotide sequencerepresented by SEQ ID NO: 2 (SEQ ID NO: 66) may be given, for example;and

[0153] (iii) as the DNA encoding a peptide consisting of an amino acidsequence which is from position 104 (Ala) to position 131 (Phe) of SEQID NO: 14 (SEQ ID NO: 67), a DNA consisting of a nucleotide sequencewhich is from position 310 to position 393 of the nucleotide sequencerepresented by SEQ ID NO: 15 (SEQ ID NO: 68) may be given, for example.

[0154] Further;

[0155] (iv) as the DNA encoding a peptide consisting of an amino acidsequence which is from position 125 (Pro) to position 131 (Phe) of SEQID NO: 1, a DNA consisting of a nucleotide sequence which is fromposition 373 to position 393 of SEQ ID NO: 2 may be given, for example;

[0156] (v) as the DNA encoding a peptide consisting of an amino acidsequence which is from position 126 (Asn) to position 131 (Phe) of SEQID NO: 1, a DNA consisting of a nucleotide sequence which is fromposition 376 to position 393 of SEQ ID NO: 2 may be given, for example;and

[0157] (vi) as the DNA encoding a peptide consisting of an amino acidsequence which is from position 127 axu) to position 131 (Phe) of SEQ IDNO: 1, a DNA consisting of a nucleotide sequence which is from position379 to position 393 of SEQ ID NO: 2 may be given, for example.

[0158] The polypeptide of the invention or RFRP-3 thereof, the receptorprotein of the invention or RFRP-3 thereof described later, and DNAsencoding these proteins and peptides may be labeled by known methods.Specifically, they may be labeled with isotopes, fluorescently labeled(e.g. with fluorescein, and so forth), biotinylated, or labeled withenzymes.

[0159] The cloning of a DNA encoding the full length of the polypeptideor the RFRP-3 of the invention (hereinafter, in the explanation of thecloning and expression of DNAs encoding these polypeptides and so forth,sometimes these peptides and so forth may be briefly referred to as thepolypeptide of the invention) can be performed either by PCRamplification from genomic DNA or cDNA using synthetic DNA primers eachhaving a partial nucleotide sequence of the polypeptide of theinvention, or by a method where a desired DNA fragment is selected byhybridizing DNA incorporated into an appropriate vector to a labeled DNAprobe, the DNA probe being a DNA fragment or a synthetic DNA encoding apart or full length of the polypeptide of the invention. Thehybridization can be carried out, for example, according to the methoddescribed in “Molecular Cloning”, 2nd Edition (J. Sambrook et al., ColdSpring Harbor Lab. Press, 1989). If commercial libraries are used, thehybridization can be carried out according to the instructions attachedthereto.

[0160] Substitution of the nucleotide sequence of a DNA can be performedby known methods such as ODA-LA PCR, the gapped duplex method, theKunkel method or modifications thereof, using known kits such asMutan™-Super Express Km (Takara), Mutan™-K (Takara) and so forth.

[0161] The cloned DNA encoding the polypeptide of the invention may beused as it is or after digestion with restriction enzymes or addition oflinkers, depending on purposes. The DNA may have ATG at its 5′ end as atranslation initiation codon and TAA, TGA, or TAG at its 3′ end as atranslation termination codon. The translation initiation andtermination codons may also be added by using appropriate synthetic DNAadapters.

[0162] Expression vectors for the polypeptide of the invention can beprepared by, for example, (a) digesting a desired DNA fragment from aDNA encoding the polypeptide of the invention and (b) ligating the DNAfragment to an appropriate expression vector downstream of its promoter.

[0163] Examples of vectors useful in the invention include plasmidsderived from Escherichia coli (e.g. pBR322, pBR325, pUC12, and pUC13);plasmids derived from Bacillus subtilis (e.g. pUB110, pTP5 and pC194);plasmids derived from yeast (e.g. pSH19 and pSH15); bacteriophages suchas λ-phage; animal viruses such as retrovirus, vaccinia virus,baculovirus; and other vectors such as pA1-11, pXT1, pRc/CMV, pRc/RSV,pcDNAI/Neo and so forth.

[0164] Any promoter may be used in the invention as long as it isappropriate for the host that will be used for expressing a desiredgene. When the host is an animal cell, examples of promoters useful inthe invention include SRα promoter, SV40 promoter, HIV LTR promoter, CMVpromoter and HSV-TK promoter.

[0165] Among these promoters, CMV (cytomegalovirus) promoter, SRαpromoter or the like is preferably used. When the host is an Escherichiabacterium, trp promoter, lac promoter, recA promoter, λ P_(L) promoter,lpp promoter, T7 promoter or the like is preferably used. When the hostis a Bacillus bacterium, SPO1 promoter, SPO2 promoter, penP promoter orthe like is preferably used. When the host is yeast, PHO5 promoter, PGKpromoter, GAP promoter, ADH promoter, or the like is preferably used.When the host is insect cell, polyhedrin promoter, P10 promoter or thelike is preferably used.

[0166] The expression vectors may, if desired, further compriseenhancers, splicing signals, polyadenylation signals, selective markers,SV40 replication origin (hereinafter, sometimes abbreviated to “SV40ori”) and the like. Examples of selective markers useful in theinvention include dihydrofolate reductase (hereinafter, sometimesabbreviated to “dhfr”) gene [methotorexate (MTX) resistance], ampicillinresistance gene (hereinafter, sometimes abbreviated to “Amp^(r)”),neomycin resistance gene [hereinafter, sometimes abbreviated to“Neo^(r)”: G418 resistance] and the like. When dhfr gene-deficientChinese hamster cells are used in combination with dhfr gene as aselective marker, a desired gene may be selected even in athymidine-free medium.

[0167] Furthermore, a signal sequence appropriate for the host may beadded, if necessary, to the N-terminal of the polypeptide of theinvention. When the host is an Escherichia bacterium, the utilizablesignal sequences may include PhoA signal sequence, OmpA signal sequenceor the like may be added. When the host is a Bacillus bacterium,α-amylase signal sequence, subtilisin signal sequence, or the like maybe added. When the host is yeast, MFα signal sequence, SUC2 signalsequence or the like may be added. When the host is animal cell, insulinsignal sequence, α-interferon signal sequence, antibody molecule signalsequence, or the like may be used.

[0168] Using the thus constructed vector comprising a DNA encoding thepolypeptide of the invention, transformants can be prepared.

[0169] Examples of hosts useful for this purpose include bacteriabelonging to the genus Escherichia, bacteria belonging to the genusBacillus, yeasts, insect cells, insects, and animal cells.

[0170] Specific examples of bacteria belonging to the genus Escherichiauseful in the invention include E. coli K12 DH1 [Proc. Natl. Acad. Sci.USA, Vol. 60, 160 (1968)], JM103 [Nucleic Acids Research, Vol. 9, 309(1981)], JA221 [Journal of Molecular Biology, Vol. 120, 517 (1978)]),HB101 [Journal of Molecular Biology, Vol, 41, 459 (1969)] and C600[Genetics, Vol. 39,440 (1954)].

[0171] Specific examples of bacteria belonging to the genus Bacillususeful in the invention include B. subtilis MI114 [Gene, Vol. 24, 255(1983)] and 207-21 [Journal of Biochemistry, Vol. 95, 87 (1984)].

[0172] Specific examples of yeasts useful in the invention includeSaccharomyces cerevisiae AH22, AH22R⁻, NA87-11A, DKD-5D and 20B-12,Schizosaccharonyces pombe NCYC1913 and NCYC2036, and Pichia pastorisKM71.

[0173] Specific examples of insect cells useful in the inventioninclude, when the virus used is AcNPV, a cell line derived from larvaeof Spodoptera fugiperda (Sf cells), MG1 cells derived from the midgut ofTrichoplusia ni, High Five™ cells derived from eggs of Trichoplusia ni,Maniestra brassicae-derived cells and Estigmena acrea-derived cells.When the virus used is BmNPV, insect cells such as a silkworm-derivedcell line (Bombyx mori N cells; BmN cells) may be used. Specificexamples of Sf cells useful in the invention include Sf9 cells (ATCC CRL1711) and Sf21 cells [both disclosed in Vaughn J. L. et al., In Vivo,13, 213-217 (1977)].

[0174] Specific examples of insects useful in the invention includelarvae of silkworm (Maeda et al., Nature, 315, 592 (1985)).

[0175] Specific examples of animal cells useful in the invention includesimian cell COS-7, Vero cells, Chinese hamster cell CHO (hereinafter,abbreviated to “CHO cells”), sdhfr gene-deficient Chinese hamster cellCHO (hereinafter, abbreviated to “CHO(dhfr⁻) cells”), mouse L cells,mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, and human FLcells.

[0176] Transformation of bacteria belonging to the genus Escherichia canbe performed in accordance with methods disclosed, for example, in Proc.Natl. Acad. Sci. USA, Vol. 69, 2110 (1972) and Gene, Vol. 17, 107(1982).

[0177] Transformation of bacteria belonging to the genus Bacillus can beperformed in accordance with methods disclosed, for example, inMolecular & General Genetics, Vol. 168, 111 (1979).

[0178] Transformation of yeasts can be performed in accordance withmethods disclosed, for example, in Methods in Enzymology, 194,182-187(1991) and Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978).

[0179] Transformation of insect cells or insects can be performed inaccordance with methods disclosed, for example, in Bio/Technology, 6,47-55 (1988).

[0180] Transformation of animal cells can be performed by methodsdisclosed, for example, in Cell Engineering, Separate Vol. 8, New CellEngineering Experiment Protocol, 263-267 (1995) (Shujunsha Co.) andVirology, Vol. 52, 456 (1973).

[0181] Thus, transformants transformed with the expression vectorcomprising a DNA encoding the polypeptide can be obtained.

[0182] As a medium to culture transformants obtained from Escherichia orBacillus bacteria as hosts, a liquid medium is appropriate. The mediummay contain carbon sources, nitrogen sources, minerals, and so on whichare necessary for the growth of the transformant. As carbon sources,glucose, dextrin, soluble starch, sucrose or the like may be enumerated.As nitrogen sources, organic or inorganic substances such as ammoniumsalts, nitrates, corn steep liquor, peptone, casein, meat extract, beancake, potato extract, or the like may be enumerated. As minerals,calcium chloride, sodium dihydrogen phosphate, magnesium chloride, orthe like may be enumerated. Further, yeast, vitamins, growth-promotingfactors, and so forth may also be added to the medium. Preferable pH ofthe medium is about 5-8.

[0183] As a medium to culture Escherichia bacteria, M9 medium containingglucose and casamino acid [Miller, Journal of Experiments in MolecularGenetics, 431433, Cold Spring Harbor Laboratory, New York, (1972)] ispreferable, for example. If necessary, medicines such as 3β-indolylacrylic acid can be added to the medium to improve the efficiency of thepromoter.

[0184] When the host is an Escherichia bacterium, the transformant iscultured usually at about 15-43° C. for about 3-24 hours. If necessary,aeration and stirring may be applied.

[0185] When the host is a Bacillus bacterium, the transformant iscultured usually at about 30-40° C. for about 6-24 hours. If necessary,aeration and stirring may also be applied.

[0186] As a medium to culture transformants obtained from yeasts ashosts, a medium such as Burkholder minimum medium [Bostian, K. L. etal., Proc. Natl. Acad. Sci. USA, Vol. 77, 4505 (1980)] or SD mediumcontaining 0.5% casamino acid [Bitter, G. A. et al., Proc. Natl. Acad.Sci. USA, Vol. 81, 5330 (1984)] may be used, for example. It ispreferable that the pH of the medium be adjusted to about 5-8. Thetransformant is cultured usually at about 20-35° C. for about 24-72hours. If necessary, aeration and stirring may be applied.

[0187] As a medium to culture transformants obtained from insect cellsor insects as hosts, Grace's Insect Medium [Grace, T. C. C., Nature,195, 788 (1962)] supplemented with additives such as inactivated 10%bovine serum may be used, for example. It is preferable that the pH ofthe medium be adjusted to about 6.26.4. The transformant is culturedusually at about 27° C. for about 3-5 days. If necessary, aeration andstirring may be applied.

[0188] As a medium to culture transformants obtained from animal cellsas hosts, examples of useful media include MEM medium [Science, Vol.122, 501 (1952)], DMEM medium [Virology, Vol. 8, 396 (1959)], RPMI 1640medium [Journal of the American Medical Association, Vol. 199, 519(1967)] and 199 medium [Proceedings of the Society of the BiologicalMedicine, Vol. 73, 1 (1950)] each containing about 5-20% fetal calfserum. Preferable pH of the medium is from about 6 to about 8. Thetransformant is cultured usually at about 30-40° C. for about 15-60hours. If necessary, aeration and stirring may be applied.

[0189] Thus, it is possible to allow the transformant to produce thepolypeptide of the invention in its cell membrane, and so forth.

[0190] Separation and purification of the polypeptide of the inventionfrom the resultant culture can be carried out, for example, according tothe methods described below.

[0191] For extraction of the polypeptide of the invention from culturedmicroorganisms or cells, the microorganism cells are harvested by knownmethods after the cultivation, suspended in a suitable buffer, anddisrupted by sonication or by lysozyme and/or freezing and thawing, andso forth. Then, a crude extract of the polypeptide extract is obtainedby centrifugation or filtration. The buffer may contain a proteindenaturing agent such as urea or guanidine hydrochloride, or asurfactant such as Triton X-100™. If the polypeptide is secreted intothe culture broth, the supernatant is separated from the microorganismsor cells after completion of the cultivation and collected by knownmethods.

[0192] Purification of the polypeptide of the invention contained in thethus obtained culture supernatant or extract can be performed by anappropriate combination of known methods for separation andpurification. These known methods include methods utilizing solubility(such as salting out or sedimentation with solvents), methods mainlyutilizing difference in molecular weight (such as dialysis,ultrafiltration, gel filtration and SDS-polyacrylamide gelelectrophoresis), methods utilizing difference in electric charge (suchas ion-exchange chromatography), methods utilizing specific affinity(such as affinity chromatography), methods utilizing difference in thehydrophobicity (such as reversed-phase high-performance liquidchromatography), and methods utilizing difference in isoelectric point(such as isoelectric electrophoresis).

[0193] When the thus obtained polypeptide of the invention is a freeform, it can be converted into the above-described salt by known methodsor methods based thereon. On the contrary, when the desired protein isobtained in a salt form, the salt can be converted into a free form oranother salt according to known methods or methods based thereon.

[0194] The polypeptide produced by the transformant can be arbitrarilymodified or a part thereof can be removed therefrom by using anappropriate protein modification enzyme before or after thepurification. Examples of such protein modification enzymes includetrypsin, chymotrypsin, arginyl endopeptidase, protein kinase andglycosidase.

[0195] The presence or the activity of the thus produced polypeptide ofthe invention or salts thereof can be measured by binding experimentsusing labeled ligands, enzyme immunoassays using specific antibodies,and so on.

[0196] As a specific example of the receptor for the polypeptide of theinvention, or amides or esters thereof, or salts thereof, or thereceptor for the RFRP-3 of the invention, or amides or esters thereof,or salts thereof (hereinafter, sometimes referred to as the “receptorprotein of the invention”), a receptor protein comprising an amino acidsequence substantially identical to the amino acid sequence representedby SEQ ID NO: 37 (OT7T022) may be given.

[0197] The receptor protein used in the invention (hereinafter, referredto as the “receptor protein of the invention”) may be a protein derivedfrom cells of any kind (e.g. splenocytes, nerve cells, glia cells,pancreatic ≢ cells, bone marrow cells, mesangial cells, Langerhan'scells, epidermal cells, epithelial cells, endothelial cells,fibroblasts, fibrous cells, muscle cells, fat cells, immune cells (e.g.macrophages, T cells, B cells, natural killer cells, mast cells,neutrophils, basophils, eosinophils, monocytes), megakaryocytes,synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts,mammary cells, hepatocytes or interstitial cells, or progenitor cells,stem cells or cancer cells of these cells and so forth) of mammals (e.g.human, guinea pig, rat, mouse, rabbit, pig, sheep, bovine, monkey and soforth) and hemocyte lineage cells or any tissue in which such cells arepresent, such as brain, various parts of brain (e.g. olfactory bulb,amygdaloid nucleus, cerebral basal nucleus, hippocampus, thalamus,hypothalamus, cerebral cortex, medulla oblongata, cerebellum, occipitallobe, frontal lobe, temporal lobe, putamen, caudate nucleus, callosum,nigra), spinal cord, pituitary gland, stomach, pancreas, kidney, liver,gonad, thyroid, gall-bladder, bone marrow, adrenal gland, skin, muscle,lung, gastrointestinal tracts (e.g. large intestine, small intestine),blood vessels, heart, thymus, spleen, submandibular gland, peripheralblood, peripheral blood cells, prostate, testis, ovary, placenta,uterus, bone, joint, skeletal muscle and so forth (especially, brain andvarious parts thereof). The receptor protein may also be a syntheticprotein.

[0198] Examples of amino acid sequences substantially identical to theamino acid sequence represented by SEQ ID NO: 37 include amino acidsequences having about 50% or more, preferably about 70% or more, morepreferably about 80% or more, still more preferably about 90% or morehomology to the amino acid sequence represented by SEQ ID NO: 37.

[0199] As a protein comprising an amino acid sequence substantiallyidentical to the amino acid sequence represented by SEQ ID NO: 37, forexample, a protein is preferable which comprises an amino acid sequencesubstantially identical to the amino acid sequence represented by SEQ IDNO: 37 and has an activity substantially the same as the activity of aprotein comprising the amino acid sequence represented by SEQ ID NO: 37.Specifically, a protein comprising the amino acid sequence representedby SEQ ID NO: 54 may be given.

[0200] Examples of substantially the same activities include ligandbinding activity or the effect of signal transduction. The term,“substantially the same” means that these activities are the naturallysame. Therefore, it is preferable that activities such as ligand bindingactivity or the effect of signal transduction should be equivalent (e.g.about 0.01- to 100 fold, preferably about 0.5- to 20-fold, morepreferably 0.5- to 2-fold). However, quantitative factors, such as thedegree of activities, the molecular weights of proteins, may bedifferent.

[0201] Activities such as ligand binding activity or the effect ofsignal transduction may be measured according to known methods. Forexample, such activities may be measured according to the liganddetermination method or screening method described later.

[0202] Further, as the receptor protein of the invention, proteinshaving the following amino acid sequences (i) to (iv) may also be used:(i) the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 54 whereinone or two or more amino acids (preferably about 1-30, more preferablyabout 1-10, and still more preferably a few (one or two) amino acids)are deleted therefrom, (ii) the amino acid sequence of SEQ ID NO: 37 orSEQ ID NO: 54 wherein one or two or more amino acids (preferably about1-30, more preferably about 1-10, and still more preferably a few (oneor two) amino acids) are added thereto, (iii) the amino acid sequence ofSEQ ID NO: 37 or SEQ ID NO: 54 wherein one or two or more amino acids(preferably about 1-30, more preferably about 1-10, and still morepreferably a few (one or two) amino acids) are substituted with otheramino acids, or (iv) an amino acid sequence which is a combination ofthe above amino acid sequences.

[0203] The receptor proteins in the present specification are expressedin accordance with the conventions for description of peptides, that is,the N-terminus (amino terminus) at the left end and the C-terminus(carboxyl terminus) at the right end. The C-terminus of the receptorprotein used in the invention may be either a carboxyl group (—COOH), acarboxylate (—COO⁻), an amide (—CONH₂) or an ester (—COOR).

[0204] Examples of R of the above ester group include C₁₋₄ alkyl groups(e.g. methyl, ethyl, n-propyl, isopropyl or n-butyl), C₃₋₈ cycloalkylgroups (e.g. cyclopentyl or cyclohexyl), C₆₋₁₂ aryl groups (e.g. phenylor α-naphthyl), C₇₋₁₄ aralkyl groups such as phenyl-C₁₋₂ alkyl groups(e.g. benzyl or phenethyl) and α-naphthyl-C₁₋₂ alkyl groups (e.g.α-naphthylmethyl). In addition, the ester group also includespivaloyloxymethyl esters that are universally used as oral esters.

[0205] When the receptor protein of the invention has a carboxyl group(or carboxylate) at any position other than its C-terminus, the carboxylgroup may be amidated or esterified; such a protein is also included inthe receptor protein of the invention. The ester in this case may be,for example, any of the esters mentioned above for the C-terminal ester.

[0206] Further, the receptor protein of the present invention alsoincludes those proteins in which the amino group of the N-terminal Metresidue is protected by a protective group (e.g. C₁₋₆ acyl group such asC₂₋₆ alkanoyl group (e.g. formyl group or acetyl group)); those proteinsin which the N-terminal Glu generated through in vivo cleavage ispyroglutaminated; those proteins in which a substituent on a side chainof an amino acid (e.g. —OH, —SH, amino group, imidazole group, indolegroup, or guannidino group) is protected by an appropriate protectivegroup (e.g. C₁₋₆ acyl group such as C₂₋₆ alkanoyl group (e.g. formylgroup or acetyl group)); and conjugated proteins such as the so-calledglycoproteins to which sugar chains are linked.

[0207] Specific examples of the receptor protein of the inventioninclude, but are not limited to, a rat-derived receptor proteinconsisting of the amino acid sequence represented by SEQ ID NO: 37 and ahuman-derived receptor protein consisting of the amino acid sequencerepresented by SEQ ID NO: 54.

[0208] The partial peptide of the receptor protein of the invention maybe any partial peptide as long as it is a partial peptide of theabove-described receptor protein. For example, a partial peptide of thereceptor protein molecule of the invention that is a site exposedoutside of the cell membrane and has receptor-binding activity may beused.

[0209] Specifically, as a partial peptide of the receptor proteincomprising the amino acid sequence represented by SEQ ID NO: 37 or SEQID NO: 54, a peptide comprising an extracellular domain (i.e.hydrophilic site) as determined by hydrophobicity plot analysis may beused. Alternatively, a peptide comprising a hydrophobic site in one ofits parts may also be used. Peptides comprising individual domainsindividually may be used; alternatively, partial peptides comprising aplurality of domains may also be use.

[0210] The number of amino acids in the partial peptide of the receptorprotein of the invention is at least 20, preferably at least 50, morepreferably at least 100 of the above-mentioned amino acid sequenceconstituting the receptor protein.

[0211] The “substantially identical” amino acid sequence means that anamino acid sequence having about 50% or more, preferably about 70% ormore, more preferably about 80% or more, still more preferably about 90%or more, most preferably about 95% or more homology to those amino acidsequences.

[0212] The term “substantially the same activity” means as definedabove. The measurement of the substantially the same activity may beperformed as described above.

[0213] Further, the partial peptide of the receptor protein of theinvention may have the above-described amino acid sequence in which oneor two or more (preferably, about 1-10, more preferably a few (one ortwo)) amino acids are deleted; or one or two or more (preferably, about1-20, more preferably about 1-10, still more preferably a few (one ortwo)) amino acids are added; or one or two or more (preferably, about1-10, more preferably a few (one or two)) amino acids are substitutedwith other amino acids.

[0214] Further, the C-terminus of the partial peptide of the receptorprotein of the invention may be either a carboxyl group (—COOH), acarboxylate (—COO⁻), an amide (—CONH₂) or an ester (—COOR) (where R isas defined above)

[0215] When the partial peptide of the receptor protein of the inventionhas a carboxyl group (or carboxylate) at any position other than itsC-terminus, the carboxyl group may be amidated or esterified; such apeptide is also included in the partial peptide of the receptor proteinof the invention. The ester in this case may be, for example, any of theesters mentioned above for the C-terminal ester.

[0216] Further, like the receptor protein described above, the partialpeptide of the receptor protein of the present invention also includesthose peptides in which the amino group of the N-terminal Met residue isprotected by a protective group; those peptides in which the N-terminalGlu generated through in vivo cleavage is pyroglutaminated; thoseproteins in which a substituent on a side chain of an amino acid isprotected by an appropriate protective group; and conjugated peptidessuch as the so-called glycopeptides to which sugar chains are linked.

[0217] As salts of the receptor protein of the invention or partialpeptides thereof, especially preferable are physiologically acceptableacid addition salts. Examples of such salts include salts formed withinorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromicacid or sulfuric acid) and salts formed with organic acids (e.g. aceticacid, formic acid, propionic acid, fumaric acid, maleic acid, succinicacid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,methanesulfonic acid or benzenesulfonic acid).

[0218] The receptor protein of the invention or salts thereof can beproduced from the afore-mentioned cells or tissues of human or othermammals by known purification methods for proteins. Alternatively, theycan also be produced by culturing a transformant comprising a DNAdescribed later encoding the receptor protein of the invention (as ahost, the same host for the transformant comprising a DNA encoding thepolypeptide of the invention may be used, for example). Such atransformant may be prepared in accordance with the procedures forpreparing the transformant comprising a DNA encoding the polypeptide ofthe invention. Alternatively, they can be produced in accordance withthe procedures for peptide synthesis which are described earlier.

[0219] When the receptor protein or salts thereof are produced fromtissues or cells of human or other mammals, the relevant tissue or cellis homogenized and then the desired protein and so forth is extractedwith acids and so forth. The desired protein and so forth can bepurified and isolated from the resultant extract by a combination ofchromatography, such as reversed phase chromatography, ion exchangechromatography and so on.

[0220] The partial peptide of the receptor protein of the invention orsalts of the partial peptide may be produced in accordance with knownmethods of peptide synthesis. Alternatively, they may be produced bydigesting the receptor protein of the invention with an appropriatepeptidase.

[0221] The receptor protein of the invention or salts thereof, and thepartial peptide of the receptor protein or amides, esters or saltsthereof may be produced in accordance with the above-describedprocedures for the synthesis of the polypeptide of the invention, oramide, esters or salts thereof.

[0222] The polynucleotide encoding the receptor protein of the inventionmay be any polynucleotide as long as it comprises a nucleotide sequence(DNA or RNA; preferably DNA) encoding the receptor protein of theinvention. The polynucleotide may be a DNA or RNA (such as mRNA)encoding the receptor protein of the invention, and may bedouble-stranded or single-stranded. When the polynucleotide isdouble-stranded, it may be a double-stranded DNA, a double-stranded RNA,or a DNA:RNA hybrid. When the polynucleotide is single-stranded, it maybe a sense strand (i.e. coding strand) or an anti-sense strand (i.e.non-coding strand).

[0223] With the polynucleotide encoding the receptor protein of theinvention, it is possible to quantitatively determine the mRNA of thereceptor protein of the invention by such methods as described in NewPCR and Its Application (Extra Issue of Experimental Medicine), 15 (7),1997 or modifications thereof.

[0224] The DNA encoding the receptor protein of the invention may be anyof the following DNAs: genomic DNA, genomic DNA library, cDNA derivedfrom the above-mentioned cells or tissues, cDNA library derived from theabove-mentioned cells or tissues, or synthetic DNA. Vectors used forlibrary construction may be any vectors such as bacteriophage, plasmid,cosmid, phagemid, and so on. Alternatively, total RNA or mRNA fractionmay be prepared from the above-mentioned cells or tissues, followed bydirect amplification by reverse transcriptase polymerase chain reaction(hereinafter, abbreviated to “RT-PCR”).

[0225] Specific examples of the DNA encoding the receptor protein of theinvention include a DNA comprising the nucleotide sequence representedby SEQ ID NO: 38, SEQ ID NO: 55 or SEQ ID NO: 56; or a DNA whichcomprises a nucleotide sequence hybridizable to the nucleotide sequencerepresented by SEQ ID NO: 38, SEQ ID NO: 55 or SEQ ID NO: 56 underhighly stringent conditions and encodes a receptor protein havingsubstantially the same activity in nature (e.g. ligand-binding activity,signal transducing effect, and so forth) as the activity of the receptorprotein of the invention.

[0226] As the DNA hybridizable to the nucleotide sequence represented bySEQ ID NO: 38, SEQ ID NO: 55 or SEQ ID NO: 56 under highly stringentconditions, a DNA which comprises a nucleotide sequence having about 70%or more, preferably about 80% or more, still more preferably about 90%or more, most preferably about 95% or more homology to the nucleotidesequence represented by SEQ ID NO: 38, SEQ ID NO: 55 or SEQ ID NO: 56may be used, for example.

[0227] The hybridization can be carried out, for example, according tothe method described in “Molecular Cloning”, 2nd Edition (J. Sambrook etal., Cold Spring Harbor Lab. Press, 1989). When commercial libraries areused, the hybridization can be carried out according to the instructionsattached thereto. Preferably, the hybridization can be carried out underhighly stringent conditions.

[0228] “Highly stringent conditions” refers to, for example, conditionswhere sodium concentration is about 19-40 mM, preferably about 19-20 mM,and temperature is about 50-70° C., preferably about 60-65° C. Inparticular, conditions where sodium concentration is about 19 mM andtemperature is about 65° C. are most preferable.

[0229] The polypeptide encoded by the DNA hybridizable to the nucleotidesequence represented by SEQ ID NO: 38, SEQ ID NO: 55 or SEQ ID NO: 56may be prepared in accordance with the above-described procedures forthe production of the polypeptide of the invention. Amides, esters andsalts of this polypeptide may be the same as the amides, esters andsalts of the polypeptide of the invention.

[0230] More specifically, as the DNA encoding the receptor proteinconsisting of the amino acid sequence represented by SEQ ID NO: 37, aDNA consisting of the nucleotide sequence represented by SEQ ID NO: 38may be used, for example. As DNA encoding the receptor proteinconsisting of the amino acid sequence represented by SEQ ID NO: 54, aDNA consisting of the nucleotide sequence represented by SEQ ID NO: 55or SEQ ID NO: 56 may be used, for example.

[0231] The expression “polynucleotide comprising a part of thenucleotide sequence of a DNA encoding the receptor protein of theinvention or a part of the nucleotide sequence complementary to the DNA”is intended to include not only the DNA encoding the partial peptide ofthe invention but also RNA encoding the same.

[0232] The DNA encoding the partial peptide of the receptor protein ofthe invention may be any DNA as long as it comprises a nucleotidesequence described above encoding the partial peptide of the invention.Further, the DNA may be any of the following DNAs: genomic DNA, genomicDNA library, cDNA derived from the above-mentioned cells or tissues,cDNA library derived from the above-mentioned cells or tissues, orsynthetic DNA. Vectors used for library construction may be any vectorssuch as bacteriophage, plasmid, cosmid, phagemid, and so on.Alternatively, mRNA fraction may be prepared from the above-mentionedcells or tissues, followed by direct amplification by reversetranscriptase polymerase chain reaction (hereinafter, abbreviated to“RT-PCR”).

[0233] Specific examples of the DNA encoding the partial peptide of theinvention include, but are not limited to, (1) a DNA comprising a partof the nucleotide sequence of a DNA comprising the nucleotide sequencerepresented by SEQ ID NO: 38, SEQ ID NO: 55 or SEQ ID NO: 56 or (2) aDNA comprising a part of the nucleotide sequence of a DNA whichcomprises a nucleotide sequence hybridizable to the nucleotide sequencerepresented by SEQ ID NO: 38, SEQ ID NO: 55 or SEQ ID NO: 56 underhighly stringent conditions and encodes a receptor protein havingsubstantially the same activity in nature (e.g. ligand-binding activity,signal transducing effect and so forth) as the activity of the receptorprotein of the invention.

[0234] The antibody against the invention to RFRP-3 (hereinafter,sometimes briefly referred to as the “antibody of the invention”) may beeither a polyclonal antibody or monoclonal antibody as long as it iscapable of recognizing the antibody of the invention against RFRP-3.

[0235] The antibody of the invention against RFRP-3 can be prepared inaccordance with known procedures for preparing antibodies or antiserausing the RFRP-3 of the invention as an antigen.

[0236] [Preparation of Monoclonal Antibodies]

[0237] (a) Preparation of Monoclonal Antibody-Producing Cells

[0238] The RFRP-3 of the invention is administered to warm-bloodedanimals either alone or together with a carrier or diluent to a sitecapable of producing antibodies upon the administration. In order toenhance the ability to produce antibodies, complete Freund's adjuvantsor incomplete Freund's adjuvants may also be administered. Theadministration is usually carried out once in every two to six weeks andtwo to ten times in the total. Examples of warm-blooded animals usefulin the invention include monkey, rabbit, dog, guinea pig, mouse, rat,sheep, goat and chicken. Among them, mouse or rat is used preferably.

[0239] In the preparation of monoclonal antibody-producing cells,individuals with detectable antibody titers are selected fromwarm-blooded animals (e.g. mice) immunized with antigen. Then, thespleen or lymph nodes are collected from them two to five days after thefinal immunization, and antibody-producing cells contained therein arefused with myeloma cells of a homologous or heterologous animal tothereby obtain monoclonal antibody-producing hybridomas. Measurement ofantibody titers in antisera may be carried out, for example, by reactinga labeled polypeptide (which will be described later) with theantiserum, followed by measuring the activity of the labeling agentbound to the antibody. The cell fusion may be carried out by a knownmethod, for example, the method of Koehler and Milstein (Nature, 256,495, (1975)). Examples of useful fusion promoters include polyethyleneglycol (PEG), Sendai virus and so forth. Preferably, PEG is used.

[0240] Examples of myeloma cells useful in the invention include myelomacells of warm-blooded animals such as NS-1, P3U1, SP2/0, AP-1 and soforth. Preferably, P3U1 is used. A preferable ratio of the number ofantibody-producing cells used (spleen cells) to the number of myelomacells is from about 1:1 to about 20:1. When PEG (preferably PEG 1000 toPEG 6000) is added at a concentration of about 10-80% and the resultantcell mixture is incubated at 20-40° C. (preferably, at 30-37° C.) forabout 1-10 minutes, an efficient cell fusion can be achieved.

[0241] Various methods may be used for screening for monoclonalantibody-producing hybridomas. For example, hybridoma culturesupernatant is added to a solid phase (e.g. microplate) on which theRFRP-3 antigen has been adsorbed either directly or with a carrier.Then, a radioactively or enzymatically labeled anti-immunoglobulinantibody (anti-mouse immunoglobulin antibody is used when mouse cellsare used in the cell fusion) or protein A is added thereto to detectmonoclonal antibodies bound to the solid phase. Alternatively, a methodmay be used in which hybridoma culture supernatant is added to a solidphase on which an anti-immunoglobulin antibody or protein A has beenabsorbed; then, a radioactively or enzymatically labeled polypeptide isadded thereto to thereby detect monoclonal antibodies bound to the solidphase.

[0242] Selection of monoclonal antibodies may be carried out by knownmethods or methods based on them. Usually, selection can be carried outin a medium for culturing animal cells supplemented with HAT(hypoxanthine, aminopterin and thymidine). As a medium for selection andculturing, any medium may be used as long as hybridomas are capable ofgrowing therein. Examples of useful media include RPMI 1640 mediumcontaining about 1-20% (preferably about 10-20%) of fetal calf serum,GIT medium (Wako Pure Chemical Industries, Ltd.) containing about 1-20%of fetal calf serum and a serum-free medium for hybridoma cultivation(SFM-101; Nissui Pharmaceutical Co.). The cultivation temperature isusually about 20-40° C., preferably about 37° C. The cultivation periodis usually from five days to three weeks, preferably one to two weeks.The cultivation may be carried out usually under 5% carbon dioxide. Theantibody titer of hybridoma culture supernatant may be measured in thesame manner as in the above-mentioned measurement of the antibody titersin antisera.

[0243] (b) Purification of the Monoclonal Antibodies

[0244] Separation and purification of monoclonal antibodies may becarried out by conventional methods, such as methods forseparating/purifying immunoglobulin [e.g. salting-out, alcoholprecipitation, isoelectric precipitation, electrophoresis,adsorption/desorption using ion exchangers (e.g. DEAE),ultracentrifugation, gel filtration, specific purification methods inwhich only an antibody is collected by means of an antigen-binding solidphase or active adsorbent such as protein A or protein G, followed bydissociation of the bond].

[0245] [Preparation of Polyclonal Antibodies]

[0246] The polyclonal antibody of the invention can be produced by knownmethods or methods based on them. For example, an immunogen (RFRP-3antigen) per se or a complex of the immunogen and a carrier protein isprepared. Then, using the immunogen or the complex, warm-blooded animalsare immunized in the same manner as described for the production ofmonoclonal antibodies. Fractions containing the antibody against thepolypeptide of the invention are harvested from the immunized animals,followed by separation and purification of the antibody.

[0247] With respect to the immunogen-carrier protein conjugate for usein the immunization of warm-blooded animals, the kind of carrier proteinand the mixing ratio of the carrier and the hapten are not particularlyrestricted as long as antibodies are produced efficiently against thehapten cross-linked to the carrier. For example, bovine serum albumin,bovine thyroglobulin, hemocyanin, or the like is coupled to the haptenat a weight ratio of about 0.1-20:1, preferably about 1-5:1.

[0248] A variety of condensing agents can be used for the couplingbetween the hapten and the carrier. For example, glutaraldehyde,carbodiimide, maleimide, or active ester reagents containing a thiol ordithiopyridyl group may be used.

[0249] The condensation product is administered to a warm-blooded animaleither alone or together with a carrier or diluent at a site capable ofproducing antibodies upon the administration. In order to enhance theantibody production ability, complete Freund's adjuvant or incompleteFreund's adjuvant may also be administered. Administration is carriedout generally once in about every 2-6 weeks and about 3-10 times in thetotal.

[0250] Polyclonal antibodies can be recovered from the blood, abdominaldropsy or other body fluid, preferably from the blood, of thewarm-blooded animal immunized as described above.

[0251] Polyclonal antibody titers in antisera can be determined in thesame manner as described above for the determination of monoclonalantibody titers in antisera. The separation and purification ofpolyclonal antibodies can be carried by the same methods for separationand purification of immunoglobulin as those described for the separationand purification of monoclonal antibodies.

[0252] With respect to the antisense DNA having a nucleotide sequencecomplementary to or substantially complementary to the DNA encoding theRFRP-3 of the invention (hereinafter, this DNA may be referred to as the“DNA of the invention”), any antisense DNA may be used as long as it hasa nucleotide sequence complementary to or substantially complementary tothe DNA of the invention and has an effect capable of inhibiting theexpression of this DNA.

[0253] A nucleotide sequence substantially complementary to the DNA ofthe invention refers to, for example, a nucleotide sequence having about70% or more, preferably about 80% or more, more preferably about 90% ormore, most preferably about 95% or more homology to the full-length or apartial nucleotide sequence of the complementary nucleotide sequence tothe DNA of the invention (i.e., the complementary strand to the DNA ofthe invention). Particularly preferable is an antisense DNA having about70% or more, preferably about 80% or more, more preferably about 90% ormore, most preferably about 95% or more homology to a part of thecomplementary strand to the DNA of the invention encoding an N-terminalregion of the RFRP-3 of the invention (e.g. nucleotide sequence encodinga region neighboring the initiation codon). These antisense DNAs can besynthesized with known DNA synthesizers.

[0254] (1) Therapeutic and/or Prophylactic Agents for Various DiseasesWhere the RFRP-3 of the Invention is Involved

[0255] The RFRP-3 of the invention, or amides or esters thereof, orsalts thereof (hereinafter, sometimes these may be briefly referred toas the “RFRP-3 of the invention”) have an effect in regulating prolactinsecretion, i.e. promoting and inhibiting effects on prolactin secretion.Since the RFRP-3 of the invention has an effect in promoting prolactinsecretion, the RFRP-3 may be used as a medicine, for example, as aprophylactic and/or therapeutic agent for various diseases associatedwith prolactin secretion failure.

[0256] On the other hand, since the RFRP-3 of the invention has highaffinity with the receptor protein of the invention, desensitizationagainst prolactin secretion will occur when administered at a high dose.As a result, the RFRP-3 of the invention also has an effect ininhibiting prolactin secretion. In such a case, the RFRp-3 can be usedas a prophylactic and/or therapeutic agent for various diseasesassociated with excessive prolactin secretion.

[0257] Therefore, as an agent for promoting prolactin secretion, theRFRP-3 of the invention is useful as a prophylactic and/or therapeuticagent for various diseases associated with prolactin secretion, such ashypoovarianism, seminal vesicle hypoplasia, osteoporosis, menopausalsyndrome, hypogalactia, hypothyroidism or renal insufficiency.

[0258] Further, since the RFRP-3 of the invention has aphrodisiac effect(pheromone-like effect) based on its effect in promoting prolactinsecretion, it is also useful as an aphrodisiac agent.

[0259] Further, as an inhibitor of prolactin secretion, the RFRP-3 ofthe invention is useful as a prophylactic and/or therapeutic agent forvarious diseases associated with prolactin secretion, such ashyperprolactinemia, pituitary adenoma, diencephalic tumor, menstrualdisorder, stresses, autoimmune diseases, prolactinoma, sterility,impotence, amenorrhea, galactorrhea, acromegaly, Chiari-Frommelsyndrome, Argonz-del Castillo syndrome, Forbes-Albright syndrome, breastcancer lymphoma, Sheehan's syndrome or spermatogenic disorder.

[0260] Further, the RFRP-3 of the invention is also useful as acontraceptive based on its prolactin secretion-inhibiting effect.

[0261] Besides, the RFRP-3 of the invention is also useful as a medicinefor testing prolactin secretion function or a veterinary medicine suchas a promoting agent for milk secretion in mammals such as bovine, goat,pig and so forth. Further, it is expected to apply the RFRP-3 to auseful substance production system where the useful substance is allowedto be produced in mammal bodies and then secreted into their milk.

[0262] Further, since the RFRP-3 of the invention has placentalfunction-regulating effect, the RFRP-3 is also useful as a prophylacticand/or therapeutic agent for choriocarcinoma, hydatidiform mole,invasive mole, miscarriage, fetal hypotrophy, glucose metabolismdisorder, lipidosis or induction of delivery.

[0263] The effect of regulating prolactin secretion of the RFRP-3 of theinvention may be measured by methods described, for example, inNeuroendocrinology, vol. 62, 1995, pp. 198-206 or Neuroscience Letters,vol. 203, 1996, pp. 164170, or modifications thereof. Preferably, themeasurement is carried out by the methods described later in Examples.

[0264] Further, since the RFRP-3 of the invention has pain-inducingeffect, the RFRP-3 is also useful as a prophylactic and/or therapeuticagent for paralgesia.

[0265] When the RFRP-3 of the invention is used as the above-describedmedicine or veterinary medicine, the RFRP-3 may be used according toconventional procedures. For example, the RFRP-3 of the invention may beadministered orally in the form of tablets (sugar-coated, if necessary),capsules, elixirs, microcapsules or the like; or parenterally in theform of injections such as aseptic solutions or suspensions in water orother pharmaceutically acceptable liquids. These preparations may beproduced, for example, by mixing the RFRP-3 of the invention or a saltthereof with physiologically acceptable carriers, flavoring agents,excipients, vehicles, antiseptics, stabilizers, binders and so forth inunit dosage forms required for preparing generally approvedpharmaceutical preparations. The amounts of active ingredients in theseformulations are decided so that an appropriate dose within thespecified range can be obtained.

[0266] Examples of additives which may be mixed in tablets, capsules andso forth include binders such as gelatin, corn starch, tragacanth andgum arabic, excipients such as crystalline cellulose, swelling agentssuch as corn starch, gelatin and alginic acid, lubricants such asmagnesium stearate, sweetening agents such as sucrose, lactose andsaccharin, and flavoring agents such as peppermint, akamono oil andcherry. When the unit dosage form is capsule, liquid carrier such asoils and fats may further be included in addition to the above-mentionedmaterials. Sterile compositions for injection can be formulatedaccording to conventional practices in pharmaceutical manufacturing,e.g., by dissolving or suspending active ingredients, naturallyoccurring vegetable oils such as sesame oil, coconut oil, and so forthin vehicles such as water for injection.

[0267] Examples of aqueous liquids for injection include physiologicalsaline and isotonic solutions containing glucose and other auxiliaryagents (e.g. D-sorbitol, D-mannitol, sodium chloride, and so forth).They may be used in combination with a suitable auxiliary solubilizersuch as alcohol (e.g. ethanol and so forth), polyalcohol (e.g. propyleneglycol, polyethylene glycol, and so forth), nonionic surfactant (e.g.Polysorbate 80™, HCO-50, and so forth). Examples of oily liquids forinjection include sesame oil, soybean oil, and so forth. They may beused in combination with an auxiliary solubilizer such as benzylbenzoate, benzyl alcohol, and so forth. In addition, buffers (e.g.phosphate buffer, sodium acetate buffer, and so forth), analgesic agents(e.g. benzalkonium chloride, procaine hydrochloride, and so forth),stabilizers (e.g. human serum albumin, polyethylene glycol, and soforth), preservatives (e.g. benzyl alcohol, phenol, and so forth),antioxidants, and so forth may also be admixed therewith. Usually, theprepared injections are filled in appropriate ampoules aseptically.

[0268] Since the thus obtained preparations are safe and of lowtoxicity, they can be administered to mammals (e.g., human, rat, mouse,guinea pig, rabbit, sheep, pig, bovine, horse, cat, dog, monkey, sacredbaboon, chimpanzee, and so forth).

[0269] Although dose levels of the RFRP-3 of the invention may varydepending on symptoms and so on, the RFRP-3 of the invention isgenerally administered to adult patients with hypothyroidism (60 kg inbody weight) at a dose of about 0.1-100 mg/day, preferably about 1.0-50mg/day, more preferably about 1.0-20 mg/day when administered orally.With respect to parenteral administration, if the RFRP-3 of theinvention is administered, for example, in the form of an injection, itis convenient to intravenously inject the RFRP-3 into adult patientswith hypothyroidism (60 kg in body weight) at a dose of about 0.01-30mg/day, preferably about 0.1-20 mg/day, and more preferably about 0.1-10mg/day, though the dose per administration may vary depending on thepatient to be treated, symptoms, method of administration, and so forth.For other animals, corresponding doses may be administered afterappropriate conversion of the above-mentioned values per 60 kg.

[0270] (2) Method of Screening for Compounds or Salts Thereof thatPromote or Inhibit the Activity of the RFRP-3 of the Invention

[0271] A method of screening for compounds or salts thereof that promoteor inhibit the activity of the RFRP-3 of the invention comprising usingthe RFRP-3 of the invention is preferably a method of screening forcompounds or salts thereof that promote or inhibit the activity of theRFRP-3 of the invention comprising using the RFRP-3 of the invention andthe receptor protein of the invention or a partial peptide, amide, esteror salt thereof (hereinafter, they are sometimes referred to as the“receptor protein of the invention”).

[0272] Specifically, this screening method is performed by measuring theactivities of the RFRP-3 of the invention in case (i) where the receptorprotein of the invention is contacted with the RFRP-3 of the inventionand in case (ii) where the receptor protein of the invention and a testcompound are contacted with the RFRP-3 of the invention, and thencomparing the results.

[0273] Specifically, the above screening method is characterized bymeasuring and comparing, for example, the cell stimulatory activities ofthe RFRP-3 of the invention and the test compound or the amounts ofbinding of the RFRP-3 of the invention and the test compound to thereceptor protein of the invention in case (i) and case (ii).

[0274] The cell stimulatory activity and so forth of the RFRP-3 of theinvention may be measured by known methods such as those described inDockray, G. J. et al., Nature, 305: 328-330, 1983; Fukusumi, S., et al.,Biochem. Biophys. Res. Commun., 232: 157-163, 1997; Hinuma, S., et al.,Nature, 393: 272-276, 1998; Tatemoto, K., et al. Biochem. Biophys. Res.Commun., 251: 471-476, 1998, or modifications thereof.

[0275] The amounts of binding of the RFRP-3 of the invention and thetest compound to the receptor protein of the invention and the cellstimulatory activities thereof may be measured by the methods describedlater or modifications thereof.

[0276] The test compound may be, for example, a peptide, protein,non-peptidic compound, synthetic compound, fermentation product, cellextract, plant extract, or animal tissue extract. These compounds may beeither novel compounds or known compounds.

[0277] For carrying out the above screening method, the RFRP-3 of theinvention is suspended in a buffer appropriate for screening to therebyprepare a sample of the RFRP-3 of the invention. Any buffer may be usedas long as it does not inhibit the reaction between the RFRP-3 of theinvention and the receptor protein of the invention; e.g. phosphatebuffer, Tris-HCl buffer and so forth of pH about 4-10 (preferably about6-8) may be used.

[0278] For example, a test compound which promotes the cell deathinhibitory activity (e.g. survival ratio) in (ii) above by about 20% ormore, preferably by about 30% or more, more preferably by about 50% ormore compared to the activity in (i) above may be selected as acompound, or a salt thereof, that promotes the activity of thepolypeptide of the invention.

[0279] For example, a test compound which increases the cell stimulatoryactivity and so forth in (ii) by about 20% or more, preferably by about30% or more, more preferably by about 50% or more compared to thecorresponding activity in (i) above may be selected as a compound thatpromotes the cell stimulatory activity and so forth of the RFRP-3 of theinvention. On the other hand, a test compound which inhibits the cellstimulatory activity and so forth in (ii) by about 20% or more,preferably by about 30% or more, more preferably by about 50% or morecompared to the corresponding activity in (i) above may be selected as acompound that inhibits the cell stimulatory activity and so forth of theRFRP-3 of the invention.

[0280] Prior to these tests, it is preferable to confirm that testcompounds bind to the receptor protein of the invention by testing thecompounds in accordance with the procedures (i) to (iii), ormodifications thereof, described in later in “Measurement of the Amountsof Binding of the RFRP-3 of the Invention and Test Compounds to theReceptor Protein of the Invention”.

[0281] Further, as an indicator for judging that the above-describedtest compound is a compound or a salt thereof that promotes or inhibitsthe activity of the RFRP-3 of the invention, there are amounts ofbinding between RFRP-3 of the present invention or the experimentalcompound and the receptor protein of the present invention and anactivity that inhibits the binding of the labeled RFRP-3 of theinvention to the receptor protein of the invention. For example, a testcompound that inhibits the binding of the labeled RFRP-3 by 10% or moreat 1×10⁻² M or below in such a binding test system as described inHosoya M. et al., Biochem. Biophys. Res. Commun. 194(1), 133-143, 1993is considered very likely to be a compound or a salt thereof thatpromotes or inhibits the activity of RFRP-3 of the invention. However,binding inhibitory activity is a relative value measured based on thebinding of the labeled RFRP-3. Therefore, binding inhibitory activity isnot an essential item for judging that the relevant test compound is acompound or a salt thereof that promotes or inhibits the activity ofRFRP-3 of the invention.

[0282] The screening kit of the invention contains the RFRP-3 of theinvention. Preferably, the screening kit of the invention furthercontains the receptor of the RFRP-3 of the invention, i.e. the receptorprotein of the invention (specifically, a protein comprising an aminoacid sequence identical or substantially identical to the amino acidsequence represented by SEQ ID NO: 37 or a salt thereof, or a partialpeptide of the protein, or an amide, ester or salt of the partialpeptide).

[0283] As one example of the screening kit of the invention, thefollowing may be given.

[0284] 1. Screening Reagents

[0285] (i) Measuring Buffer and Washing Buffer

[0286] Hanks' Balanced Salt Solution (Gibco) supplemented with 0.05%bovine serum albumin (Sigma).

[0287] This buffer may be stored at 4° C. after sterilization with afilter 0.45 μm in pore size or may be prepared at the time of use.

[0288] (ii) Receptor Sample

[0289] CHO cells expressing the receptor protein of the invention aresub-cultured to 12-well plates at 5×10⁵ cells/well and then cultured for2 days at 37° C., under 5% CO₂ 95% air.

[0290] (iii) Labeled Ligand

[0291] An aqueous solution of the RFRP-3 of the invention labeled with acommercial radioisotope such as [³H], [¹²⁵I], [¹⁴C] or [³⁵S] is storedat 4° C. or −20° C. This solution is diluted to 1 μM with the measuringbuffer at the time of use.

[0292] (iv) Ligand Standard Solution

[0293] The RFRp-3 of the invention is dissolved in 0.1% bovine serumalbumin (Sigma)-containing PBS to give a concentration of 1 mM andstored at −20° C.

[0294] 2. Measuring Method

[0295] (i) The CHO cells expressing the receptor protein of theinvention cultured in 12-well tissue culture plates are washed twicewith 1 ml of the washing buffer. Then, 490 μl of the measuring buffer isadded to each well.

[0296] (ii) After addition of 5 μl of a test compound (10⁻³-10⁻¹⁰ M), 5μl of the labeled ligand is added and reacted for 1 hr at roomtemperature. In order to know the amount of non-specific binding, 5 μlof 10⁻³ M ligand is added instead of the test compound.

[0297] (iii) The reaction solution is removed, and the cells are washedthree times with 1 ml of the washing buffer. The labeled ligand bound tothe cells is dissolved with 0.2 N NaOH-1% SDS and mixed with 4 ml ofLiquid Scintillator A (Wako Purechemical Industries).

[0298] (iii) The radioactivity is measured with a liquid scintillationcounter (Beckman). Then, percent maximum binding (PMB) is determinedwith the numerical formula described below.

[0299] [Numerical Formula I]

PMB=[(B−NSB)/(B ₀ −NSB)]×100

[0300] where

[0301] PMB: percent maximum binding

[0302] B: value when a test compound is added

[0303] NSB: amount of non-specific binding

[0304] B₀: amount of maximum binding

[0305] The compounds or salts thereof obtainable by using the screeningmethod or screening kit of the invention are compounds that are selectedfrom the above-described test compounds (such as peptides, proteins,non-peptidic compounds, synthetic compounds, fermentation products, cellextracts, plant extracts, animal tissue extracts, plasma and so forth)and promote or inhibit the activity (e.g. cell stimulatory activity) ofthe RFRP-3 of the invention.

[0306] As salts of such compounds, the same salts as described earlieron the salts of the RFRP-3 of the invention may be used.

[0307] (3) Methods of Measuring the Amounts of Binding of the RFRP-3 ofthe Invention and Test Compounds to the Receptor Protein of theInvention and the Cell Stimulatory Activities Thereof.

[0308] By using the receptor protein of the invention or constructing anexpression system for a recombinant receptor protein and using areceptor binding assay system with the expression system, it is possibleto measure the amounts of binding and the cell stimulatory activities ofthose compounds (e.g. peptides, protein, non-peptidic compounds,synthetic compounds, fermentation products, and so forth) that bind tothe receptor protein of the invention and have cell stimulatory activity(e.g. activity to promote or inhibit the liberation of arachidonic acid,the liberation of acetylcholine, intracellular Ca²⁺ liberation,intracellular cAMP production, intracellular cGMP production, inositolphosphate production, changes in cell membrane potentials, thephosphorylation of intracellular proteins, the activation of c-fos, thelowering of pH, and so forth).

[0309] The measuring methods are characterized by measuring, forexample, the amounts of binding of test compounds to the receptorprotein of the invention or the cell stimulatory activities of the testcompounds when the receptor protein of the invention is contacted withthe RFRP-3 of the invention or the test compounds.

[0310] More specifically, the present invention provides the followingmeasuring methods:

[0311] (i) a measuring method wherein a labeled RFRP-3 of the inventionor a labeled test compound is contacted with the receptor protein of theinvention, and then the amount of binding of the labeled RFRP-3 or thelabeled test compound to the receptor protein is measured;

[0312] (ii) a measuring method wherein a labeled RFRP-3 of the inventionor a labeled test compound is contacted with cells, or a membranefraction thereof, containing the receptor protein of the invention, andthen the amount of binding of the labeled RFRP-3 or the labeled testcompound to those cells or the membrane fraction is measured;

[0313] (iii) a measuring method wherein a labeled RFRP-3 of theinvention or a labeled test compound is contacted with a receptorprotein expressed on cell membranes which has been prepared by culturinga transformant comprising a DNA encoding the receptor protein of theinvention, and then the amount of binding of the labeled RFRP-3 or thelabeled test compound to the receptor protein is measured;

[0314] (iv) a measuring method wherein a labeled RFRP-3 of the inventionor a labeled test compound is contacted with cells containing thereceptor protein of the invention, and then the cell stimulatoryactivities mediated by the receptor protein (e.g. activity to promote orinhibit the liberation of arachidonic acid, the liberation ofacetylcholine, intracellular Ca²⁺ liberation, intracellular cAMPproduction, intracellular cGMP production, inositol phosphateproduction, changes in cell membrane potentials, the phosphorylation ofintracellular proteins, the activation of c-fos, the lowering of pH andso forth) are measured; and

[0315] (v) a measuring method wherein a labeled RFRP-3 of the inventionor a labeled test compound is contacted with a receptor proteinexpressed on cell membranes which has been prepared by culturing atransformant comprising a DNA encoding the receptor protein of theinvention, and then the cell stimulatory activities mediated by thereceptor protein (e.g. activity to promote or inhibit the liberation ofarachidonic acid, the liberation of acetylcholine, intracellular Ca²⁺liberation, intracellular cAMP production, intracellular cGMPproduction, inositol phosphate production, changes in cell membranepotentials, the phosphorylation of intracellular proteins, theactivation of c-fos, the lowering of pH and so forth) are measured.

[0316] It is especially preferable to conduct the tests described in (i)to (iii) above to confirm that the test compound binds to the receptorprotein of the invention, and then to conduct the tests described in(iv) and (v) above.

[0317] First, the receptor protein used in the measuring method may beany material as long as it contains the afore-mentioned receptor proteinof the invention. A suitable example is a receptor protein expressed ina large quantity in animal cells.

[0318] For the production of the receptor protein of the invention, theafore-mentioned expression method may be used. Preferably, the receptorprotein is produced by expressing a DNA encoding the receptor protein inmammalian cells or in insect cells. As a DNA fragment encoding thedesired protein portion, usually a complementary DNA is used though theDNA is not limited to a complementary DNA. For example, a gene fragmentor synthetic DNA may also be used. In order to introduce the DNAfragment encoding the receptor protein of the invention into a hostanimal cell and to express it efficiently, it is preferred that the DNAfragment should be incorporated downstream of a promoter such aspolyhedron promoter derived from nuclear polyhedrosis virus belonging tobaculovirus, promoter derived from SV40, promoter derived fromretrovirus, metallothionein promoter, human heat shock promoter,cytomegalovirus promoter, SR a promoter and so forth. The quantity andthe quality of the expressed receptor can be examined by known methods.For example, methods described in Nambi, P. et al: The Journal ofBiochemical Society, vol.267, pp. 19555-19559 (1992) may be used.

[0319] Thus, in the above measuring method, the material containing thereceptor protein of the invention may be either the receptor protein ofthe invention purified according to known methods, or cells or amembrane fraction thereof containing the receptor protein.

[0320] When cells containing the receptor protein of the invention areused in the measuring method, the cells may be fixed in glutaraldehyde,formalin and so forth. Such fixation may be carried out according toknown methods.

[0321] “Cells containing the receptor protein of the invention” refersto host cells expressing the receptor protein of the invention. Examplesof the host cell useful in the invention include Escherichia coli,Bacillus subtilis, yeasts, insect cells and animal cells.

[0322] The “cell membrane fraction” refers to a cell membrane-richfraction which is obtainable by known methods after disruption of cells.Examples of methods of cell disruption include a method of squeezingcells using a Potter-Elvehjem homogenizer, disruption by a Waringblender or a Polytron (Kinematica), disruption by ultrasonication,disruption via ejection of cells from small nozzles while applying apressure using a French press or the like and so forth. For thefractionation of cell membranes, centrifugal fractionation such asdifferential centrifugation or density gradient centrifugation is mainlyused. For example, disrupted cells are centrifuged at a low speed (500rpm to 3,000 rpm) for a short period (usually, from about one to tenmin); the resultant supernatant is further centrifuged at a high speed(15,000 rpm to 30,000 rpm) usually for 30 min to 2 hr. and the resultingprecipitate is used as a membrane fraction. The membrane fractioncontains large quantities of the expressed receptor protein andcell-derived membrane components such as phospholipids and membraneproteins.

[0323] The amount of the receptor protein in the receptorprotein-containing cells or the membrane fraction thereof is preferably10³ to 10⁸ molecules per cell, more preferably 10⁵ to 10⁷ molecules percell. Incidentally, the greater the expressed amount, the higher theligand binding activity (specific activity) per membrane fractionwhereby the construction of a highly sensitive screening system becomespossible and, moreover, it permits measurement of a large amount ofsamples with the one same lot.

[0324] For conducting the above-mentioned methods (i) to (iii), anappropriate receptor protein fraction and a labeled test compound arenecessary.

[0325] As the receptor protein fraction, a natural receptor proteinfraction or a recombinant protein fraction having an activity equivalentto that of the natural protein fraction is preferable. The term“activity equivalent to” refers to equivalent ligand binding activity,signal transducing effect, or the like.

[0326] As the labeled test compound, a compound selected from peptides,proteins, non-peptidic compounds, synthetic compounds, fermentationproducts, cell extracts, plant extracts, animal tissue extracts, plasma,and so forth and labeled with [³H], [¹²⁵I], [¹⁴C], [³⁵S] and so forth.

[0327] Specifically, first, cells or cell membrane fractions containingthe receptor protein of the invention are suspended in a buffer suitablefor the assay to thereby prepare the receptor sample. The buffer may beany buffer, such as Tris-HCL buffer or phosphate buffer with pH 4-10(preferably pH 6-8), as long as it does not inhibit the binding of theligand to the receptor protein. In order to reduce non-specific binding,surfactants such as CHAPS, Tween 80™ (Kao-Atlas), digitonin ordeoxycholate and various proteins such as bovine serum albumin (BSA) orgelatin may be added to the buffer. Further, in order to inhibit thedegradation of the receptor and the ligand by protease, a proteaseinhibitor such as PMSF, leupeptin, E-64 (Peptide Institute) or pepstatinmay be added. A specific amount (5,000 cpm to 500,000 cpm) of a testcompound labeled with [³H], [¹²⁵I], [¹⁴C], [³⁵S] or the like is allowedto co-exist in 0.01 ml-10 ml of the receptor solution. In order to knowthe amount of non-specific binding (NSB), a reaction tube to which agreatly excessive amount of the unlabeled test compound has been addedis also prepared. The reaction is carried out at 0-50° C., preferably at4-37° C. for about 20 min to 24 hr, preferably about 30 min to 3 hr.After the reaction, the reaction solution is filtered through a glassfiber filter or the like, and washed with the appropriate amount of thesame buffer. Then, the radioactivity remaining in the glass fiber filteris measured with a liquid scintillation counter or a gamma-counter. Whenthe count (B-NSB) obtained by subtracting the non-specific bindingamount (NSB) from the total binding amount (B) is more than 0 cpm, thetest compound can be selected as a compound that promotes the activityof the RFRP-3 of the invention.

[0328] In order to conduct the methods described in (iv) and (v) above,the cell stimulatory activity mediated by the receptor protein (e.g.activity to promote or inhibit the liberation of arachidonic acid, theliberation of acetylcholine, intracellular Ca²⁺ liberation,intracellular cAMP production, intracellular cGMP production, inositolphosphate production, changes in cell membrane potentials, thephosphorylation of intracellular proteins, the activation of c-fos, thelowering of pH, and so forth.) may be measured by known methods or withcommercial measuring kits. Specifically, first, cells containing thereceptor protein are cultured in multi-well plates or the like. Themedium is exchanged with a fresh medium or an appropriate buffer withoutcytotoxicity. Then, the RFRP-3 of the invention or a test compound isadded to the cells, which are incubated for a specific period. Afterextraction of the cells or recovery of the supernatant, the resultantproducts are determined quantitatively according to respectiveprocedures. When it is difficult to assay the production of a substanceto be used as an indicator of cell stimulatory activity (e.g.arachidonic acid) due to a protease contained in the cells, the assaymay be performed with the addition of an inhibitor to the protease. Withrespect to such activity as inhibition of cAMP production, the activitymay be detected as a production inhibitory effect upon those cells whichwere treated with forskolin or the like to increase their basic yield.

[0329] The measuring kit described above contains the receptor proteinof the invention, cells containing the receptor protein of theinvention, or a cell membrane fraction of cells containing the receptorprotein of the invention.

[0330] As one example of such a measuring kit, the following may begiven.

[0331] 1. Screening Reagents

[0332] (i) Measuring Buffer and Washing Buffer

[0333] Hanks' Balanced Salt Solution (Gibco) supplemented with 0.05%bovine serum albumin (Sigma).

[0334] This buffer may be stored at 4° C. after sterilization with afilter 0.45 μm in pore size or may be prepared at the time of use.

[0335] (ii) G Protein-Coupled Receptor Protein Sample

[0336] CHO cells expressing the receptor protein of the invention aresub-cultured to 12-well plates at 5×10⁵ cells/well and then cultured for2 days at 37° C. under 5% CO₂ 95% air.

[0337] (iii) Labeled Test Compound

[0338] A compound labeled with a commercial radioisotope such as [³H],[¹²⁵I], [¹⁴C] or [³⁵S], or a compound labeled with other appropriatemethod. An aqueous solution of the labeled test compound is stored at 4°C. or −20° C. and diluted to 1 μM with the measuring buffer at the timeof use. Those test compounds which are sparingly soluble in water, theyare dissolved in dimethyl formamide, DMSO, methanol, or the like.

[0339] (iv) Unlabeled Test Compound

[0340] The same compound used for the labeled test compound is preparedat a concentration 100- to 1000-fold higher than that of the labeledtest compound.

[0341] 2. Measuring Method

[0342] (i) The CHO cells expressing the receptor protein of theinvention cultured in 12-well tissue culture plates are washed twicewith 1 ml of the washing buffer. Then, 490 μl of the measuring buffer isadded to each well.

[0343] (ii) Five μl of the labeled test compound is added to each welland reacted for 1 hr at room temperature. In order to know the amount ofnon-specific binding, 5 μl of the unlabeled test compound is added inadvance.

[0344] (iii) The reaction solution is removed, and the cells are washedthree times with 1 ml of the washing buffer. The labeled test compoundbound to the cells is dissolved with 0.2 N NaOH-1% SDS and mixed with 4ml of liquid Scintillator A (Wako Purechemical Industries).

[0345] (iii) The radioactivity is measured with a liquid scintillationcounter (Beckman).

[0346] (4) Medicines Comprising the Compounds Obtained by the ScreeningMethod of the Invention

[0347] The compound or salts thereof obtained by using the screeningmethod or the screening kit of the invention may be used as aprophylactic and therapeutic agent for various diseased associated withinsufficient prolactin secretion when the compound has an effect ofpromoting prolactin secretion; and the compound or salts thereof may beused as a prophylactic and/or therapeutic agent for various diseasesassociated with excessive prolactin secretion when the compound has aneffect of inhibiting prolactin secretion.

[0348] When the obtained compound or salts thereof have an effect ofpromoting prolactin secretion, they are useful as a prophylactic and/ortherapeutic agent for various diseases associated with prolactinsecretion, such as hypoovarianism, seminal vesicle hypoplasia,osteoporosis, menopausal syndrome, hypogalactia, hypothyroidism or renalinsufficiency as an agent for promoting prolactin secretion.

[0349] Further, since the compound or salts thereof have aphrodisiaceffect (pheromone-like effect) based on their effect in promotingprolactin secretion, they are also useful as an aphrodisiac agent.

[0350] On the other hand, when the obtained compound or salts thereofhave an effect in inhibiting prolactin secretion, they may be used as aprophylactic and/or therapeutic agent for various diseases associatedwith excessive prolactin secretion. The compound or salts thereof areuseful as a prophylactic and/or therapeutic agent for various diseasesassociated with prolactin secretion, such as hyperprolactinemia,pituitary adenoma, diencephalic tumor, menstrual disorder, stresses,autoimmune diseases, prolactinoma, sterility, impotence, amenorrhea,galactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-del Castillosyndrome, Forbes-Albright syndrome, breast cancer lymphoma or Sheehan'ssyndrome or spermatogenic disorder, as an inhibitor of prolactinsecretion.

[0351] Further, the compound or salts thereof are also useful as acontraceptive based on their effect in inhibiting prolactin secretion.

[0352] Besides, the obtained compound or salts thereof are also usefulas a medicine for testing the function of prolactin secretion orveterinary medicine such as a promoting agent for milk secretion inmammals such as bovine, goat or pig. Further, it is expected to applysuch compound or salts thereof to a production system for a usefulsubstance where the useful substance is allowed to be produced in mammalbodies and then secreted into their milk.

[0353] Further, since the obtained compound or salts thereof haveplacental function-regulating effect, they are also useful as aprophylactic and/or therapeutic agent for choriocarcinoma, hydatidiformmole, invasive mole, miscarriage, fetal hypotrophy, glucose metabolismdisorder, lipidosis or induction of delivery.

[0354] The effect in regulating prolactin secretion of the compound orsalts thereof obtained by using the above-described screening method orscreening kit may be measured by methods described, for example, inNeuroendocrinology, vol. 62, 1995, pp. 198-206 or Neuroscience Letters,vol. 203, 1996, pp. 164-170, or modifications thereof. Preferably, themeasurement is carried out by the methods described later in Examples.

[0355] Further, compounds that promote the activity of the RFRP-3 of theinvention are useful as a prophylactic and/or therapeutic agent forparalgesia.

[0356] On the other hands, compounds that inhibit the activity of theRFRP-3 of the invention are useful as an analgesic agent.

[0357] When the obtained compound or a salt thereof is used as theabove-described medicine or veterinary medicine, they may be usedaccording to conventional procedures. For example, the compounds orsalts thereof may be administered orally in the form of tablets(sugar-coated, if necessary), capsules, elixirs, microcapsules or thelike; or parenterally in the form of injections such as asepticsolutions or suspensions in water or other pharmaceutically acceptableliquids. These preparations may be produced, for example, by mixing thecompound or a salt thereof with physiologically acceptable carriers,flavoring agents, excipients, vehicles, antiseptics, stabilizers,binders, and so forth in unit dosage forms required for preparinggenerally approved pharmaceutical preparations. The amounts of activeingredients in these formulations are decided so that an appropriatedose within the specified range can be obtained.

[0358] Examples of additives which may be mixed in tablets, capsules,and so forth include binders such as gelatin, corn starch, tragacanthand gum arabic, excipients such as crystalline cellulose, swellingagents such as corn starch, gelatin and alginic acid, lubricants such asmagnesium stearate, sweetening agents such as sucrose, lactose andsaccharin, and flavoring agents such as peppermint, akamono oil andcherry. When the unit dosage form is capsule, liquid carrier such asoils and fats may further be included in addition to the above-mentionedmaterials. Sterile compositions for injection can be formulatedaccording to conventional practices in pharmaceutical manufacturing,e.g., by dissolving or suspending active ingredients, naturallyoccurring vegetable oils such as sesame oil, coconut oil, and so forthin vehicles such as water for injection.

[0359] Examples of aqueous liquids for injection include physiologicalsaline and isotonic solutions containing glucose and other auxiliaryagents (e.g. D-sorbitol, D-mannitol, sodium chloride, and so forth).They may be used in combination with a suitable auxiliary solubilizersuch as alcohol (e.g. ethanol, and so forth.), polyalcohol (e.g.propylene glycol, polyethylene glycol, and so forth), nonionicsurfactant (e.g. Polysorbate 80™, HCO-50, and so forth). Examples ofoily liquids for injection include sesame oil, soybean oil, and soforth. They may be used in combination with an auxiliary solubilizersuch as benzyl benzoate, benzyl alcohol, and so forth. In addition,buffers (e.g. phosphate buffer, sodium acetate buffer, and so forth),analgesic agents (e.g. benzalkonium chloride, procaine hydrochloride,and so forth), stabilizers (e.g. human serum albumin, polyethyleneglycol, and so forth), preservatives (e.g. benzyl alcohol, phenol, andso forth), antioxidants, and so forth may also be admixed therewith.Usually, the prepared injections are filled in appropriate ampoulesaseptically.

[0360] Since the thus obtained preparations are safe and of lowtoxicity, they can be administered to mammals (e.g., human, rat, mouse,guinea pig, rabbit, sheep, pig, bovine, horse, cat, dog, monkey, sacredbaboon, chimpanzee, and so forth).

[0361] Although dose levels of the obtained compound or a salt thereofmay vary depending on symptoms and so on, the compound or a salt thereofis generally administered to adult patients with hypothyroidism (60 kgin body weight) at a dose of about 0.1-100 mg/day, preferably about1.0-50 mg/day, more preferably about 1.0-20 mg/day when administeredorally. With respect to parenteral administration, if the compound or asalt thereof is administered, for example, in the form of an injection,it is convenient to inject intravenously to adult patients withhypothyroidism (60 kg in body weight) at a dose of about 0.01-30 mg/day,preferably about 0.1-20 mg/day, and more preferably about 0.1-10 mg/day,though the dose per administration may vary depending on the patient tobe treated, symptoms, method of administration, and so forth. For otheranimals, corresponding doses may be administered after appropriateconversion of the above-mentioned values per 60 kg.

[0362] (5) Quantitative Determination of RFRP-3 Using the Antibody ofthe Invention

[0363] Since the antibody of the invention can specifically recognizethe RFRP-3 of the invention, the antibody may be used for quantitativedetermination of the RFRP-3 of the invention contained in samplesolutions, in particular, in quantitative determination by sandwichimmnunoassay.

[0364] The present invention provides:

[0365] (i) a method of quantitative determination of the RFRP-3 of theinvention in a sample solution, comprising competitively reacting theantibody of the invention with the sample solution and the RFRP-3 of theinvention labeled, and determining the ratio of the labeled RFRP-3 ofthe invention bound to the antibody; and

[0366] (ii) a method of quantitative determination of the RFRP-3 of theinvention in a sample solution, comprising reacting the sample solutionwith the antibody of the invention insolubilized on a carrier andanother antibody of the invention labeled, simultaneously or insuccession, and determining the activity of the labeling agent on theinsolubilized carrier.

[0367] In the quantitative determination method described in (ii) above,it is preferred that one antibody should be an antibody that recognizesan N-terminal region of the RFRP-3 of the invention and the otherantibody should be an antibody that reacts with a C-terminal region ofthe RFRP-3 of the invention.

[0368] Further, the monoclonal antibody of the invention may be used toquantitatively determine the RFRP-3 of the invention or may be used fordetection of the RFRP-3 by tissue staining. For these purposes, eitherantibody molecules per se or the F(ab′)₂, Fab′ or Fab fragment thereofmay be used.

[0369] Methods of quantitative determination of the RFRP-3 of theinvention using the antibody of the invention are not particularlylimited. Any measuring method may be used in which the amount ofantibody, antigen or antibody-antigen complex corresponding to theamount of the antigen in a sample solution (e.g. the amount of theRFRP-3 of the invention) is detected by chemical or physical means, andthen calculated from a standard curve prepared using a standard solutioncontaining a known amount of the antigen. For example, nephrometry,competitive methods, immunometric methods and sandwich assay may be usedconveniently and, in terms of sensitivity and specificity, the sandwichassay described later is particularly preferred.

[0370] Examples of labeling agents useful in measuring methods utilizinglabeling substances include radioisotopes, enzymes, fluorescentsubstances, and luminescent substances. Examples of radioisotopesinclude [¹²⁵I], [¹³¹I], [³] and [¹⁴C]. Preferred examples of enzymes arethose which are stable and with high specific activity, e.g.,β-galactosidase, β-glucosidase, alkali phosphatase, peroxidase andmalate dehydrogenase. Examples of fluorescent substances includefluorescamine and fluorescein isothiocyanate. Examples of luminescentsubstances include luminol, luminol derivatives, luciferin, andlucigenin. Further, a biotin-avidin system may also be used for bindingan antibody or antigen with a labeling agent.

[0371] Insolubilization of antigens or antibodies may be performed byphysical adsorption or by chemical binding usually used forinsolubilizing or immobilizing peptides or enzymes. Examples of carriersuseful for this purpose include insoluble polysaccharides such asagarose, dextran and cellulose; synthetic resins such as polystyrene,polyacrylamide and silicone; and glass.

[0372] In the sandwich assay, a sample solution is reacted with aninsolubilized monoclonal antibody of the invention (primary reaction);then, another monoclonal antibody of the invention that is labeled isreacted therewith (secondary reaction); and the activity of the labelingagent on the insolubilzed carrier is measured to thereby quantitativelydetermine the amount of the RFRP-3 of the invention in the samplesolution. The primary reaction and the secondary reaction may beconducted in the reverse order, or they may be conducted simultaneouslyor with an interval. The type of the labeling agent and the method ofinsolubilization may be the same as those described herein earlier. Inimmunoassays using the sandwich technique, the antibody insolubilized ona solid phase or the antibody labeled is not necessarily a singleantibody, a mixture of two or more antibodies may be used for thepurposes of enhancing the sensitivity of measurement, and so forth.

[0373] In the method of measuring the RFRP-3 of the invention by thesandwich assay of the invention, the monoclonal antibodies of theinvention used in the primary and the secondary reactions are preferablythose antibodies whose sites of binding to the RFRP-3 of the inventionare different from each other. For example, if the antibody used in thesecondary reaction recognizes a C-terminal region of the RFRP-3 of theinvention, an antibody that recognizes a site other than the C-terminalregion, e.g. an N-terminal region, is preferably used in the primaryreaction.

[0374] The monoclonal antibody of the invention may be used in ameasuring system other than the sandwich assay, such as competitivemethods, immunometric methods and nephrometry.

[0375] In competitive methods, an antigen in a sample solution and alabeled antigen are reacted competitively with an antibody, then,unreacted labeled antigen (F) and labeled antigen bound to the antibody(B) are separated (i.e. B/F separation); and the amount of the label ofB or F is measured to thereby quantitatively determine the amount of theantigen in the sample solution. With respect to this reaction method,there are a liquid phase method in which a soluble antibody is used, andthe B/F separation is conducted with polyethylene glycol, and a secondantibody to the above-mentioned antibody is used; and a solid phasemethod in which a solidified antibody is used as the first antibody or asoluble antibody is used as the first antibody while a solidifiedantibody is used as the second antibody.

[0376] In immunometric methods, an antigen in a sample solution and asolidified antigen are reacted competitively with a specific amount of alabeled antibody, followed by separation of the solid phase from theliquid phase; or an antigen in a sample solution is reacted with anexcessive amount of a labeled antibody, and then a solidified antigen isadded to bind unreacted labeled antibody to the solid phase, followed byseparation of the solid phase from the liquid phase. Subsequently, theamount of label in one of the phases is measured to determine the amountof the antigen in the sample solution.

[0377] In nephrometry, the amount of insoluble precipitate generated asa result of antigen-antibody reaction in a gel or solution is measured.Even when the amount of the antigen in a sample solution is small andthus only a small amount of such precipitate is obtained, lasernephrometry utilizing the scattering of laser can be used conveniently.

[0378] In applying each of those immunological measuring methods to themeasuring method of the present invention, no special conditions oroperations are required. A measuring system for the polypeptide of thepresent invention may be constructed using the conventional conditionsand operational procedures in the relevant measuring method while takinginto account usual technical consideration of those skilled in the art.For details of these commonly used technical means, a variety ofreviews, reference books, and so forth may be referred to.

[0379] For example, Hiroshi Irie (ed.): “Radioimmunoassay” (Kodansha,1974); Hiroshi Irie (ed.): “Radioimmunoassay; Second Series” (Kodansha,1979), Eiji Ishikawa et al. (ed.): “Enzyme Immunoassay” (Igaku Shoin,Japan, 1978); Eiji Ishikawa et al. (ed.): “Enzyme Immunoassay” (SecondEdition) (Igaku Shoin, 1982); Eiji Ishikawa et al. (ed.): “EnzymeImmunoassay” (Third Edition) (Igaku Shoin, 1987); “Methods inEnzymology”, Vol. 70 (Immunochemical Techniques (Part A)); ibid., Vol.73 (Immunochemical Techniques (Part B)); ibid., Vol. 74 (ImmunochemicalTechniques (Part C)); ibid., Vol. 84 (Immunochemical Techniques (Part D:Selected Immunoassays)); ibid., Vol. 92 (Immunochemical Techniques (PartE: Monoclonal Antibodies and General Immunoassay Methods)); ibid., Vol.121 (Immunochemical Techniques (Part I: Hybridoma Technology andMonoclonal Antibodies)) (Academic Press) and the like may be referredto.

[0380] By using the antibody of the invention as described above, theRFRP-3 of the invention can be quantitatively determined with highsensitivity.

[0381] Further, when abnormality is detected in the concentration of theRFRP-3 of the invention in a subject by quantitatively determining theconcentration of the RFRP-3 of the invention using the antibody of theinvention, it is possible to diagnose that the subject has one of thefollowing diseases associated with prolactin secretion, such ashypoovarianism, seminal vesicle hypoplasia, osteoporosis, menopausalsyndrome, hypogalactia, hypothyroidism, renal insufficiency,hyperprolactinemia, pituitary adenoma, diencephalic tumor, menstrualdisorder, stresses, autoimmune diseases, prolactinoma, sterility,impotence, amenorrhea, galactorrhea, acromegaly, Chiari-Frommelsyndrome, Argonz-del Castillo syndrome, Forbes-Albright syndrome, breastcancer lymphoma, Sheehan's syndrome or spermatogenic disorder, orparalgesia; or that the subject is very likely to develop such a diseasein the future.

[0382] Further, the antibody of the invention may be used for detectingthe RFRP-3 of the invention present in body fluids, tissues or othersamples. The antibody of the invention may also be used in thepreparation of antibody columns for use in the purification of theRFRP-3 of the invention; in the detection of the RFRP-3 of the inventionin individual fractions generated in the course of purification; and inthe analysis of the behavior of the RFRP-3 of the invention in testcells.

[0383] (6) Gene Diagnostics

[0384] The DNA of the invention can, when used as a probe for example,detect abnormalities in DNA or mRNA encoding the RFRP-3 of the invention(gene abnormalities) in human or other warm-blooded animals (e.g. rat,mouse, guinea pig, rabbit, bird, sheep, pig, bovine, horse, cat, dog,monkey, and so forth). Thus, the DNA of the invention is useful as agene diagnostic for diagnosing, e.g., damage, mutations or reducedexpression of the above DNA or mRNA, or increase or excessive expressionof the above DNA or mRNA.

[0385] Gene diagnosis using the DNA of the invention may be performed byknown methods such as Northern hybridization or PCR-SSCP method(Genomics, Vol. 5, 874879 (1989); Proceedings of the National Academy ofSciences of the USA, 86: 2766-2770 (1989)).

[0386] When abnormality in expression is detected by Northernhybridization, for example, it is possible to diagnose that the relevantsubject is very likely to have one of the following diseases associatedwith prolactin secretion, such as hypoovarianism, seminal vesiclehypoplasia, osteoporosis, menopausal syndrome, hypogalactia,hypothyroidism, renal insufficiency, hyperprolactinemia, pituitaryadenoma, diencephalic tumor, menstrual disorder, stresses, autoimmunediseases, prolactinoma, sterility, impotence, amenorrhea, galactorrhea,acromegaly, Chiari-Frommel syndrome, Argonzdel Castillo syndrome,Forbes-Albright syndrome, breast cancer lymphoma, Sheehan's syndrome orspermatogenic disorder, or paralgesia; or that the subject is verylikely to develop such a disease in the future.

[0387] (7) Medicines Comprising Antisense DNA

[0388] Antisense DNA that complementarily binds to the DNA of theinvention and thus inhibits the expression of that DNA is useful as aprophylactic and/or therapeutic agent for the above-described variousdiseases associated with prolactin secretion or as an analgesic agent.

[0389] For example, when the antisense DNA is used, the antisense DNAalone or the antisense DNA inserted into an appropriate vector such as aretrovirus vector, adenovirus vector, adeno-associated virus vector, andso forth may be administered using conventional means. The antisense DNAmay be administered as it is or after formulation with physiologicallyacceptable carriers such as adjuvants to promote uptake, by means of agene gun or a catheter such as hydrogel catheter. Formulation may beperformed in the same manner as the formulation of medicines comprisingthe RFRP-3 of the invention.

[0390] Dose levels of the antisense DNA of the invention may varydepending upon the patient to be treated, the target organ, symptoms,administration route, and so on. In oral administration, generally, theantisense DNA is administered to patients with sterility (60 kg in bodyweight) at a dose of about 0.1-100 mg/day, preferably about 1.0-50mg/day, more preferably about 1.0-20 mg/day. In parenteraladministration, when the antisense DNA of the invention is administered,for example, in the form of an injection, it is convenient to inject theantisense DNA of the invention intravenously to patients withhyperprolactinemia (60 kg in body weight) at a dose of about 0.01-30mg/day, preferably about 0.1-20 mg/day, more preferably about 0.1-10mg/day, though the dose level may vary depending on the patient to betreated, the target organ, symptoms, administration route, and so on.For other animals, corresponding doses may be administered afterappropriate conversion of the above-mentioned values per 60 kg.

[0391] Further, the antisense DNA may be used as an oligonucleotideprobe for diagnostic purposes to examine the presence or state ofexpression of the DNA of the invention in tissues or cells.

[0392] (8) Medicines Comprising the Antibody of the Invention

[0393] The antibody of the invention that has an effect of neutralizingthe activity of the RFRP-3 of the invention is useful as a prophylacticand/or therapeutic agent for the above-described diseases associatedwith prolactin secretion or as an analgesic agent.

[0394] The above-mentioned prophylactic and/or therapeutic agentscomprising the antibody of the invention may be administered orally orparenterally to human or other mammals (e.g. rat, rabbit, sheep, pig,bovine, cat, dog, monkey, and so forth) in the forms of liquidpreparations without any processing or in appropriate forms ofpharmaceutical compositions. Dose levels may vary depending upon thepatient to be treated, the target disease, symptoms, administrationroute, and so on. However, it is convenient to inject the antibody ofthe invention intravenously at a dose of about 0.01-20 mg/kg bodyweight, preferably about 0.1-10 mg/cg body weight, more preferably about0.1-5 mg/kg body weight per administration about one to five times aday, preferably about one to three times a day. In other parenteraladministration and oral administration, similar dose levels may be used.If symptoms are particularly heavy, the dose may be increasedaccordingly.

[0395] The antibody of the invention may be administered alone or in theforms of appropriate pharmaceutical compositions. The pharmaceuticalcompositions for the above administration comprise the antibody or saltthereof, pharmacologically acceptable carriers, and diluents orexcipients. Such compositions are provided in forms appropriate for oralor parenteral administration.

[0396] For example, compositions for oral administration include solidor liquid preparations such as tablets (including sugar-coated tabletsand film-coated tablets), pills, granules, dispersants, capsules(including soft capsules), syrups, emulsions and suspensions. Thesecompositions are prepared according to conventional methods and containcarriers, diluents or excipients conventionally used in the field ofmedicine manufacturing. For example, lactose, starch, sucrose, magnesiumstearate and the like are used as carriers or excipients for tablets.

[0397] Compositions for parenteral administration include, for example,injections and suppositories. Injections include intravenous injections,subcutaneous injections, intradermal injections, muscle injections,instilment injections, and so forth. Such injections may be prepared bydissolving, suspending or emulsifying the above antibody or salt thereofin an aseptic, aqueous or oily liquid. Examples of aqueous liquids forinjection include physiological saline and isotonic solutions containingglucose and other auxiliary agents. They may be used in combination witha suitable auxiliary solubilizer such as alcohol (e.g. ethanol),polyalcohol (e.g. propylene glycol, polyethylene glycol), nonionicsurfactant [e.g. Polysorbate 80™, HCO-50 (polyoxyethylene (50 mol)adduct of hydrogenated castor oil)], and so forth). Examples of oilyliquids for injection include sesame oil and soybean oil. They may beused in combination with an auxiliary solubilizer such as benzylbenzoate, benzyl alcohol, and so forth. Usually, the prepared injectionsare filled in appropriate ampoules. Suppositories for administrationinto rectum may be prepared by mixing the antibody or a salt thereofwith a conventional suppository base.

[0398] It is convenient to formulate the above-described pharmaceuticalcompositions for oral or parenteral administration into unit dosageforms that would give an appropriate dose of the active ingredient.Examples of such unit dosage forms include tablets, pills, capsules,injections (ampoules), and suppositories. Usually, each unit of thesedosage forms contains preferably about 5-500 mg of the above-describedantibody. In particular, each unit contains preferably about 5-100 mg ininjections, and each unit in other dosage forms contains preferablyabout 10-250 mg.

[0399] The above-described pharmaceutical compositions may contain otheractive ingredients as long as they do not produce undesirableinteraction with the above-described antibody.

[0400] (9) DNA-Transferred Animals

[0401] The present invention further provides non-human mammalsharboring a foreign DNA encoding the RFRP-3 of the invention(hereinafter referred to briefly as the “foreign DNA of the invention”)or a mutant thereof (sometimes referred to briefly as the “foreignmutant DNA of the invention”).

[0402] Thus, the present invention provides:

[0403] (1) A non-human mammal harboring the foreign DNA of the inventionor a mutant DNA thereof:

[0404] (2) The non-human mammal according to (1) which is a rodent:

[0405] (3) The non-human mammal according to (2) wherein the rodent ismouse or rat; and

[0406] (4) A recombinant vector containing the foreign DNA of theinvention or a mutant DNA thereof and capable of expressing the DNA in amammal.

[0407] The non-human mammal harboring the foreign DNA of the inventionor a mutant DNA thereof (hereinafter referred to briefly as the“DNA-transferred animal of the invention”) can be created bytransferring the desired DNA to a germinal cell such as unfertilized eggcells, fertilized egg cells, or sperm cells or primordial cells thereof,preferably during the period of embryogenesis in the ontogenesis of thenon-human mammal (more preferably, in the stage of a single cell or afertilized egg cell and generally at the 8-ell stage or earlier), by thecalcium phosphate method, electric pulse method, lipofection method,agglutination method, microinjection method, particle gun method, orDEAE-dextran method. It is also possible to transfer the foreign DNA ofthe invention of interest into somatic cells, organs in the living body,tissue cells, or the like by such DNA transfer methods to use theresultant cells or tissues in cell culture or tissue culture. Further,by fusing the resultant cells with the above-mentioned germinal cell byknown cell fusion methods, it is also possible to create theDNA-transferred animal of the invention.

[0408] The non-human mammal useful in the invention includes bovine,pig, sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse, rat, andso on. From the viewpoint of construction of diseased animal models,rodents which have comparatively short ontogenesis and life cycles andcan be easily bred, particularly mouse (e.g. pure strains such asC57BL/6, DBA2, and so forth and hybrid strains such as B6C3F₁, BDF₁,B6D2F₁, BALB/c, ICR, and so forth) or rat (e.g. Wistar, SD, and soforth), are preferred.

[0409] Examples of the “mammal” in the expression “a recombinant vectorcapable of expressing the DNA in a mammal” include human in addition tothe above-mentioned non-human mammals.

[0410] The foreign DNA of the invention is not a DNA of the inventionwhich is inherently possessed by the non-human mammal, but a DNA of theinvention that has been once isolated/extracted from a mammal.

[0411] Examples of the mutant DNAs of the invention include not only theDNAs that have variations (e.g. mutations) in the nucleotide sequence ofthe original DNA of the invention as a result of, for example, additionor deletion of nucleotides or substitution with other nucleotides, butalso abnormal DNAs.

[0412] The term “abnormal DNA” as used herein means any DNA that causesexpression of an abnormal RFRP-3 of the invention. For example, a DNAthat allows expression of an RFRP-3 that inhibits the function of thenormal RFRP-3 of the invention may be used.

[0413] The foreign DNA of the invention may be derived from a mammalthat is of the same species as that of the host animal or of differentspecies. For transfer of the DNA of the invention to the host animal, itis generally advantageous to use a DNA construct in which the DNA isligated downstream of a promoter capable of expressing the DNA in animalcells. For example, in transferring the human DNA of the invention, thishuman DNA of the invention may be ligated downstream of a promotercapable of directing expression of DNAs derived from various animals(e.g. rabbit, dog, cat, guinea pig, hamster, rat, mouse, and so forth)harboring the DNA of the invention having high homology to the human DNAto thereby prepare a DNA construct (e.g. vector), which can then bemicroinjected into fertilized egg cells of a host mammal such asfertilized mouse egg cells. Thus, a DNA-transferred mammal showing highexpression of the DNA of the invention can be created.

[0414] Examples of the expression vector for the RFRP-3 of the inventioninclude plasmids derived from E. coli, plasmids derived from B.subtilis, plasmids derived from yeast, λ phage and other bacteriophages,retroviruses such as Molony leukemia virus, and animal viruses such asvaccinia virus and vaculovirus. Preferable examples are E. coli-derivedplasmids, B. subtilis-derived plasmids and yeast-derived plasmids.

[0415] Examples of promoters that regulate the expression of the DNAinclude (1) promoters for DNAs derived from viruses (e.g. simian virus,cytomegalovirus, Molony leukemia virus, JC virus, papilloma virus,poliovirus, and so forth), (2) promoters derived from mammals (e.g.human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, and so forth),for example, promoters of albumin, insulin II, uroprakin II, elastase,erythropoietin, endothelin, muscle creatine kinase, glial fibrillaryacidic protein, glutathione S-transferase, platelet-derived growthfactor β, keratin K1, K10, and K14, collagen type I and type II, cyclicAMP-dependent polypeptide kinase βI subunit, dystrophin, tartaricacid-resistant alkaline phosphatase, atrial natriuretic factor,endothelial receptor tyrosine kinase (generally abbreviated to Tie2),sodium/potassium-dependent adenosinetriphosphatase (Na, K-ATPase),neurofilament light chain, metallothionein I and IIA, metalloproteinaseI tissue inhibitor, MHC Class I antigen (H-2L), H-ras, renin, dopamineβ-hydroxylase, thyroid peroxidase (TPO), polypeptide chain elongationfactor 1α (EF-1α), β-actin, α- and β-myosin heavy chain, myosin lightchains 1 and 2, myelin basic protein, thyroglobulin, Thy-1,immunoglobulin H chain variable region (VNP), serum amyloid P component,myoglobin, troponin C, smooth muscle α-actin, preproenkephalin A,vasopressin, and so on. Preferable are those promoters which can directhigh expression of the DNA in the whole body, e.g. cytomegaloviruspromoter, human polypeptide chain elongation factor 1α (EF-1α) promoter,and human and chicken β-actin promoters.

[0416] It is preferable that the vector has a sequence for terminatingthe transcription of the desired messenger RNA (generally calledterminator) in the DNA-transferred mammal. For example, sequencesderived from viruses or various mammals may be used. Preferably, SV40terminator derived from simian virus or the like is used.

[0417] In addition, for enhancing the expression of the desired DNAfurther, it is possible, depending on the specific purpose, to ligate asplicing signal, an enhancer domain, a portion of an eucaryotic DNAintron, and so forth upstream of the 5′-end of the promoter region,between the promoter region and the translated region, or downstream ofthe 3′-end of the translated region.

[0418] The translated region for the normal RFRP-3 of the invention maybe obtained as the entire genomic DNA or a part thereof from liver-,kidney-, thyroid cell- or fibroblast-derived DNA from human or othermammals (e.g. rabbit, dog, cat, guinea pig, hamster, rat, mouse, and soforth) or commercial genomic DNA libraries. Alternatively, thetranslated region may be obtained using, as a raw material, cDNAprepared from liver-, kidney-, thyroid cell- or fibroblast-derived DNAby conventional methods. The foreign abnormal DNA is capable of creatinga mutated translated region from the translated region for the normalRFRP-3 obtained from the above-mentioned cell or tissue by pointmutagenesis.

[0419] The translated region can be prepared as a DNA construct whichcan be expressed in a DNA-transferred animal, by conventionalrecombinant DNA techniques, i.e. by ligating it downstream of thepromoter and, if desired, upstream of the transcription terminationsite.

[0420] The transfer of the foreign DNA of the invention at thefertilized egg cell stage insures that the DNA will be ubiquitous in allthe germ cells and somatic cells of the host mammal. The presence of theDNA of the invention in the germ cells of the DNA-transferred animalfollowing DNA transfer means that all the germinal cells and somaticcells of all the subsequent generations of the DNA-transferred animalharbor the DNA of the invention. Thus, the progeny of suchDNA-transferred animal which inherited the foreign DNA of the inventionhave the DNA in all of their germ cells and somatic cells.

[0421] The non-human mammal into which the foreign normal DNA of theinvention has been transferred can be verified by mating to retain theforeign DNA stably and then bred as a line harboring that DNA fromgeneration to generation under usual breeding conditions.

[0422] The transfer of the foreign DNA of the invention at thefertilized egg cell stage insures that the DNA will be present in excessin all the germ cells and somatic cells of the host mammal. The presenceof the foreign DNA of the invention in the germ cells of theDNA-transferred animal following the DNA transfer means that all thegerminal cells and somatic cells of all the progeny of theDNA-transferred animal harbor the foreign DNA of the invention inexcess. Thus, the progeny of such DNA-transferred animal which inheritedthe foreign DNA of the invention have the DNA in excess in their germcells and somatic cells.

[0423] By preparing homozygous animals having the transferred DNA inboth homologous chromosomes and mating male animals with female animals,it is possible to breed through generations so that every progenyharbors the DNA in excess.

[0424] The non-human mammal harboring the normal DNA of the inventionfeatures a high expression of the normal DNA and may eventually developa hyperergasia of the RFRP-3 of the invention through activation of thefunction of the endogenous normal DNA. Thus, the animal can be utilizedas an animal model of that disease. For example, by using theDNA-transferred animal harboring the normal DNA of the invention, it ispossible to study the hyperergasia of the RFRP-3 of the invention, toelucidate the mechanisms of diseases with which the RFRP-3 of theinvention is associated, and to explore therapeutic modalities for thediseases.

[0425] Furthermore, the mammal to which the foreign normal DNA of theinvention has been transferred presents symptoms due to an increase inthe free RFRP-3 of the invention and, therefore, can also be used in thescreening of therapeutic medicines for diseases with which the RFRP-3 ofthe invention is associated.

[0426] On the other hand, the non-human mammal harboring the foreignabnormal DNA of the invention can be verified by mating to retain theDNA stably and then bred as a line harboring the DNA from generation togeneration under usual breeding conditions. Moreover, it is possible toincorporate the desired foreign DNA in the above-mentioned plasmid anduse it as a starting material. The DNA construct with the promoter canbe prepared by conventional recombinant DNA techniques. Transfer of theabnormal DNA of the invention in the fertilized egg cell stage insuresthat the transferred DNA will be ubiquitous in all the germ cells andsomatic cells of the host mammal. The presence of the abnormal DNA ofthe invention in the germ cells of the DNA-transferred animal means thatall the progeny of this DNA-transferred animal harbor the abnormal DNAof the invention in all of their germinal cells and somatic cells. Theprogeny of this animal harbor the abnormal DNA of the invention in allof their germinal cells and somatic cells. By preparing homozygous maleand female animals having the introduced DNA in both homologouschromosomes and mating them, it can be insured that every progenyharbors the DNA from generation to generation.

[0427] The non-human mammal harboring the abnormal DNA of the inventionfeatures a high expression of the abnormal DNA and, therefore, mayeventually develop adiaphoria associated with functional inactivation ofthe RFRP-3 of the invention through inhibition of the function of theendogenous normal DNA. Thus, the animal can be utilized as an animalmodel of that disease. For example, by using the DNA-transferred animalharboring the abnormal DNA of the invention, analysis of the mechanismof this functional inactivation adiaphoria attributable to the RFRP-3 ofthe invention and therapeutic modalities for the disease can beexplored.

[0428] As a specific potential use, the DNA-transferred animal with ahigh expression of the abnormal DNA of the invention can be used as amodel for elucidating the functional inhibition of the normalpolypeptide by the abnormal polypeptide of the invention (dominantnegative effect) in adiaphoria of functional inactivation.

[0429] Moreover, the DNA-transferred mammal harboring the foreignabnormal DNA of the invention develops symptoms due to an increase inthe free polypeptide of the invention and, therefore, can be utilized inthe screening of therapeutic medicines for adiaphoria attributable tofunctional inactivation of the polypeptide of the invention.

[0430] As other potential uses of the two types of DNA-transferredanimals harboring the two kinds of DNAs of the invention, the followingmay be considered:

[0431] (1) Use as a cell source for tissue culture;

[0432] (2) Analysis of those polypeptides which are expressed oractivated specifically by the RFRP-3 of the invention, by analyzing theDNA or RNA in tissues of the DNA-transferred animal of the invention orby analyzing the compositions of the peptides expressed by the DNA;

[0433] (3) Study of the functions of cells of those tissues which aregenerally difficult to culture, by using the cells from the tissuescontaining the DNA as cultured by the standard tissue culture technique;

[0434] (4) Screening for medicines capable of enhancing the cellfunctions by using the cells described in (3); and

[0435] (5) Isolation and purification of the mutant RFRP-3 of theinvention and construction of antibodies thereto.

[0436] Furthermore, by using the DNA-transferred animal of theinvention, clinical symptoms of diseases associated with the RFRP-3 ofthe invention, inclusive of above-described adiaphoria associated withfunctional inactivation of the RFRP-3 of the invention, can beinvestigated. In addition, more detailed pathological findings can beobtained in various organs of this model of diseases associated with theRFRP-3 of the invention, thus contributing to the development of newtherapies as well as the study and treatment of secondary diseasesarising from such diseases.

[0437] Moreover, by removing various organs from the DNA-transferredanimal of the invention, mincing them and digesting them with aproteolytic enzyme such as trypsin, free single cells harboring thetransferred DNA can be recovered. These cells can be cultured forestablishment of a cell line. Furthermore, characterization of cellsproducing the RFRP-3 of the invention can be made and their relationshipwith apoptosis, differentiation, or proliferation, the mechanism ofsignal transduction in them, and abnormalities involved can be exploredto thereby generate information useful for further elucidation of theRFRP-3 of the invention and its effects.

[0438] Moreover, for the development of therapeutic medicines fordiseases associated with the RFRP-3 of the invention, such as adiaphoriaresulted from functional inactivation of the RFRP-3 of the invention byusing the DNA-transferred animal of the invention, an effective andrapid screening technology for such therapeutic medicines can beestablished by using the test and assay methods described hereinbefore.In addition, by using the above DNA-transferred animal or the foreignDNA expression vector of the invention, gene therapies for diseasesassociated with the RFRP-3 of the invention can be explored anddeveloped.

[0439] (10) Knockout Animals

[0440] The invention further provides non-human mammalian embryonic stemcells wherein the DNA of the invention is inactivated, and non-humanmammals deficient in expression of the DNA of the invention wherein theDNA of the invention is deactivated.

[0441] The invention, therefore, provides:

[0442] (1) A non-human mammalian embryonic stem cell wherein the DNA ofthe invention is inactivated;

[0443] (2) The embryonic stem cell according to in (1) wherein the DNAis inactivated by introduction of a reporter gene (e.g. E. coli-derivedβ-galactosidase gene);

[0444] (3) The embryonic stem cell according to (1) which isneomycin-resistant;

[0445] (4) The embryonic stem cell according to (1) wherein thenon-human mammal is a rodent;

[0446] (5) The embryonic stem cell according to (4) wherein the rodentis mouse;

[0447] (6) A non-human mammal deficient in expression of the DNA of theinvention, wherein the DNA is inactivated;

[0448] (7) The non-human mammal according to (6) wherein the DNA isinactivated by introduction of a reporter gene (e.g. E. coli-derivedβ-galactosidase gene) and the reporter gene can be expressed under thecontrol of the promoter for the DNA of the invention;

[0449] (8) The non-human mammal according to (6) wherein the non-humanmammal is a rodent;

[0450] (9) The non-human mammal according to (8) wherein the rodent ismouse; and

[0451] (10) A method for screening for compounds, or salts thereof, thatenhance or inhibit the promoter activity for the DNA of the invention,which comprises administering a test compound to the non-human mammalaccording to (7) and detecting expression of the reporter gene.

[0452] The expression “non-human mammalian embryonic stem cell whereinthe DNA of the invention is inactivated” means the embryonic stem cell(hereinafter referred to briefly as ES cell) of a non-human mammal inwhich the DNA has been deprived of the capacity to express the RFRP-3 ofthe invention (hereinafter, sometimes referred to as the “knockout DNAof the invention”) through introduction of an artificial mutation to theDNA of the invention possessed by the non-human mammal to therebyinhibit expression of the DNA of the invention or through substantialdeprivation of the activity of the RFRP-3 of the invention encoded bythe DNA.

[0453] As the non-human mammals, the same animals as mentionedhereinbefore may be used.

[0454] Examples of the method for introducing an artificial mutation tothe DNA of the invention are a deletion of some or all of the DNAsequence, or an insertion of a different DNA, or substitution with adifferent DNA by the genetic engineering technology. The knockout DNA ofthe invention may be created by such a mutation that would shift thereading frame or destroy the function of the promoter or exon.

[0455] The non-human mammalian embryonic stem cell wherein the DNA ofthe invention is inactivated (hereinafter referred to as the “DNAinactivated ES cell of the invention” or the “knockout ES cell of theinvention”) can be prepared by, for example, procedures which compriseisolating the DNA of the invention from a desired non-human mammal,inserting a medicine-resistance gene, typically neomycin-resistance geneor hygromycin-resistance gene, or a reporter gene such as lacZ(β-galactosidase gene) or cat (chloramphenicol acetyltransferase gene)into its exon region to disrupt the function of the exon or inserting aDNA sequence for terminating gene transcription (e.g. poly A additionsignal) in an intron region between exons to thereby inhibit synthesisof a complete mRNA, introducing the thus-constructed DNA strand having aDNA sequence designed to eventually disrupt the gene (hereinafter,referred to briefly as the “targeting vector”) into the chromosomes ofthe host animal by homologous recombination, subjecting the resulting EScell to Southern hybridization analysis using a DNA sequence located onthe DNA of the invention or in its vicinity as a probe or a PCRprocedure using a DNA sequence located on the targeting vector and a DNAsequence in the vicinity but not including the DNA of the invention usedin the construction of the targeting vector as primers, and selectingthe knockout ES cell of the invention.

[0456] The original ES cell to be used for inactivation of the DNA ofthe invention by the homologous recombination technique or the like maybe an already established cell line such as those mentioned hereinbeforeor a new cell line established de novo by the known method of Evans andKaufman. Taking mouse ES cells as an example, ES cells of the 129 lineare generally employed but the immunological background of this line isnot clear. Therefore, the cell line established by using BDF₁ micecreated by the hybridization of C57BL/6 mice and C57BL/6 mice, bothyielding few eggs, with DBA/2 mice (BDF₁=F₁ of C57BL/6 and DBA/2) forpreparing pure-line ES cells with an immunologically defined geneticbackground can be used with advantage. In addition to the advantage ofhigh egg output and sturdiness of the egg, BDF₁ mice have the backgroundof C57BL/6 mice so that in the construction of a disease model with EScells obtained, the genetic background of the model mice can beconverted to that of C57BL/6 mice by back-crossing with C57BL/6.

[0457] Moreover, in establishing an ES cell line, it is common practiceto use blastocytes 3.5 days following fertilization but, aside fromthem, a large number of early embryos can be prepared with highefficiency by harvesting the embryos at the 8-cell stage and culturingthem into blastocytes.

[0458] Furthermore, while ES cells from both male and female animals canbe used, generally ES cells of male animals are more convenient for theconstruction of reproduction line chimeras. Moreover, for the purpose ofreducing the burden of the complicated cultural procedure, it ispreferable to carry out sexing as early as possible.

[0459] As a typical method for sexing ES cells, there can be mentionedthe method in which the gene in the sex determination region on the Ychromosome is amplified and detected by PCR. Whereas the conventionalkaryotype analysis requires about 10⁶ cells, the above method requiresonly about one colony equivalent of ES cells (about 50 cells).Therefore, the primary selection of ES cells in an early stage can bemade by this sexing method. Since male cells can thus be selected in theearly stage, the trouble in the initial stage of culture can bedrastically reduced.

[0460] Moreover, the secondary selection can be carried out by G-bandingfor the number of chromosomes. The number of chromosomes in theresulting ES cell is preferably 100% of the normal number but this goalmay not be reached due to the physical and other factors involved in theestablishment of the line. In such cases, it is preferable to knockoutthe gene of the ES cell and re-clone it into the normal cell (taking amouse as an example, the cell in which the number of chromosomes is2n=40).

[0461] The embryonic stem cell line thus established is generally verysatisfactory in proliferation characteristic but since it is liable tolose its ontogenic ability, it must be subcultured with sufficient care.For example, this cell line should be cultured on suitable feeder cellssuch as STO fibroblasts in the presence of LIF (1-10000 U/ml) in acarbon dioxide incubator (preferably 5% CO₂, 95% air or 5% oxygen, 5%CO₂, 90% air) at about 37° C. and, in subculture, it should be treatedwith trypsin/EDTA solution (generally 0.001-0.5% trypsin/0.1-5 mM EDTA,preferably about 0.1% trypsin/1 mM EDTA) to provide single cells andseed them on freshly prepared feeder cells. While such subculture isgenerally performed every 1-3 days, it is good practice to observe thecells on each occasion and, whenever morphologically abnormal cells arediscovered, discard the culture.

[0462] ES cells can be allowed to differentiate into various types ofcells, such as head long muscle cells, visceral muscle cells, heartmuscle cells, and so forth by conducting monolayer culture to a highdensity under suitable conditions or suspension culture until a mass ofcells is formed (M. J. Evans & M. H. Kaufman, Nature, 292, 154, 1981; G.R. Martin, Proceedings of National Academy of Science USA, 78, 7634,1981; T. C. Doetschman et al., Journal of Embryology and ExperimentalMorphology, 87, 27, 1985), and the cell deficient in expression of theDNA of the invention as obtainable by causing the ES cell of theinvention to differentiate is useful for the cytobiological in vitrostudy of the RFRP-3 of the invention.

[0463] The non-human mammal deficient in expression of the DNA of theinvention can be differentiated from normal animals by assaying the mRNAin the animals by the known method and comparing the amounts ofexpression indirectly.

[0464] As the non-human mammal used for this purpose, the same animalsas mentioned hereinbefore may be used.

[0465] With respect to the non-human mammal deficient in expression ofthe DNA of the invention, the DNA of the invention can be knocked out byintroducing the targeting vector constructed as described above into,for example, mouse embryonic stem cells or mouse egg cells and therebyallowing the DNA sequence of the targeting vector harboring theinactivated DNA of the invention to undergo homologous recombinationwith, and accordingly replacing, the DNA of the invention on the mouseembryonic stem cell or egg cell chromosomes.

[0466] The cell with the DNA of the invention thus knocked out can bejudged by Southern hybridization analysis using a DNA sequence on theDNA of the invention or in its vicinity as a probe or by PCR using a DNAsequence on the targeting vector or a mouse-derived DNA sequence in aregion adjacent to but not including the DNA of the invention used inthe targeting vector as primers. When a non-human mammalian embryonicstem cell is used, a cell line with the DNA of the invention knocked outby the homologous recombination technique is cloned and injected intothe non-human mammalian embryo or blastocyte at a suitable stage ofembryogenesis, for example at the 8-cell stage, and the resultingchimera embryo is transplanted in the pseudopregnant uterus of thenon-human mammal. The animal thus obtained is a chimera animalconstituted by both the cells harboring the normal DNA locus of theinvention and the cells harboring the artificially mutated DNA locus ofthe invention.

[0467] When some of the gametes of this chimera animal harbor themutated DNA locus of the invention, an individual whose entire tissue isconstituted by cells harboring the mutated DNA locus of the inventioncan be screened from the colony of animals obtained by crossing such achimera animal with a normal animal, for example by coat colordiscrimination. The individuals thus selected are usually animalshetero-deficient in expression of the RFRP-3 of the invention and bymating such individuals hetero-deficient in expression of the RFRP-3 ofthe invention with each other, animals homo-deficient in expression ofthe RFRP-3 of the invention can be acquired.

[0468] When egg cells are used, a transgenic non-human mammal with thetargeting vector having been introduced into its chromosomes can beprepared by injecting the DNA solution into the egg cell nucleus by themicroinjection technique and selecting animals expressing a mutation ofthe DNA of the invention by homologous recombination.

[0469] The individuals with the DNA of the invention thus knocked outare mated to verify that the animals obtained by mating also have theDNA knocked out and they can be sub-bred under the usual breedingconditions.

[0470] Preparation and maintenance of the germ line may also be carriedout in accordance with conventional methods. Thus, by mating male andfemale animals harboring the inactivated DNA, homozygotes having theinactivated DNA in both homologous chromosomes can be obtained. Thehomozygotes thus obtained are bred under such condition that, withregard to the dam, the number of homozygotes is plural per normalindividual. By mating male and female heterozygotes, homozygotes andheterozygotes both harboring the inactivated DNA can be sub-bread.

[0471] The non-human mammalian embryonic stem cell harboring theinactivated DNA of the invention is very useful for the construction ofnon-human mammals deficient in expression of the DNA of the invention.

[0472] Moreover, the mammal deficient in expression of the DNA of theinvention lacks various biological activities inducible by the RFRP-3 ofthe invention or the receptor protein of the invention and can,therefore, be of use as an animal model of diseases arising frominactivation of the biological activities of the RFRP-3 of the inventionor the receptor protein of the invention, thus being useful in theetiological studies of such diseases and development of therapeuticmethods.

[0473] (10a) Method of Screening for Compounds withTherapeutic/Prophylactic Effect Upon Diseases Resulted from Deficiencyor Damage of the DNA of the Invention

[0474] Non-human mammals deficient in expression of the DNA of theinvention may be used for screening for compounds with a therapeuticand/or prophylactic effect upon diseases resulted from deficiency ordamage of the DNA of the invention.

[0475] The present invention provides a method of screening forcompounds, or salts thereof, having a therapeutic and/or prophylacticeffect upon diseases resulted from deficiency or damage of the DNA ofthe invention, which is characterized by administering a test compoundto a non-human mammal deficient in expression of the DNA of theinvention and observing and measuring the changes in the mammal.

[0476] As the non-human mammal deficient in expression of the DNA of theinvention used in the above screening method, the same animals asdescribed earlier may be used.

[0477] The test compound may be, for example, a peptide, protein,non-peptidic compound, synthetic compound, fermentation product, cellextract, plant extract, animal tissue extract or plasma. These compoundsmay be either novel compounds or known compounds.

[0478] Specifically, a non-human mammal deficient in expression of theDNA of the invention is treated with a test compound and then comparedwith a control animal not treated with the compound. Subsequently, thetherapeutic and/or prophylactic effect of the test compound may beexamined using the changes in individual organs, tissues or diseasesymptoms in the mammal as indicators.

[0479] As a method for treating a test animal with a test compound, oraladministration, intravenous injection, or the like may be used. Themethod may be appropriately selected depending on the symptoms of thetest animal, the nature of the test compound, and so on. Dose levels ofthe test compound may be appropriately selected taking into account ofthe administration method, the nature of the test compound, and so on.Further, dose levels of the test compound may be appropriately selecteddepending on the administration method, the nature of the relevant testcompound, and so on.

[0480] When compounds that have a therapeutic and/or prophylactic effectupon diseases associated with insufficient prolactin secretion (e.g.hypoovarianism, seminal vesicle hypoplasia, osteoporosis, menopausalsyndrome, hypogalactia, hypothyroidism or renal insufficiency), diseasesassociated with excessive prolactin secretion (e.g. hyperprolactinemia,pituitary adenoma, diencephalic tumor, menstrual disorder, stresses,autoimmune diseases, prolactinoma, sterility, impotence, amenorrhea,galactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-del Castillosyndrome, Forbes-Albright syndrome, breast cancer lymphoma or Sheehan'ssyndrome or spermatogenic disorder), paralgesia, or the like is screenedfor, test compounds are administered to non-human mammals deficient inthe expression of the DNA of the invention, followed by measurement ofthe time course of blood prolactin levels, and so forth in the mammals.

[0481] In the above screening method, when a test compound has beenadministered to a test animal and, as a result, the amount of prolactinsecretion of the test animal has changed by about 10% or more,preferably by about 30% or more, more preferably by about 50% or more,the test compound can be selected as a compound that has a therapeuticand/or prophylactic effect upon the above-described diseases.

[0482] Compounds obtainable by using the screening method of theinvention are compounds selected from the above-mentioned test compoundsand have a therapeutic and/or prophylactic effect upon diseases resultedfrom deficiency or damage of the RFRP-3 of the invention. Therefore,they may be used as medicines that are safe and of low toxicity, such asprophylactic and/or therapeutic agents for those diseases that are safeand of low toxicity. Furthermore, compounds derived from those compoundsobtained by the above screening may also be used in the same manner.

[0483] The compound obtained by the above screening may be in a saltform. As salts of the compounds, salts formed with physiologicallyacceptable acids (e.g. organic or inorganic acids) or bases (e.g. alkalimetals) may be used. Especially preferable are physiologicallyacceptable acid addition salts. Examples of such salts include saltsformed with inorganic acids (e.g. hydrochloric acid, phosphoric acid,hydrobromic acid or sulfuric acid) and salts formed with organic acids(e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleicacid, succinic acid, tartaric acid, citric acid, malic acid, oxalicacid, benzoic acid, methanesulfonic acid or benzenesulfonic acid).

[0484] Medicines comprising the compound, or a salt thereof, obtained bythe above screening may be prepared in the same manner as described formedicines comprising the RFRP-3 of the invention.

[0485] Since the thus obtained preparations are safe and of lowtoxicity, they may be administered to, for example, human and othermammals (such as rat, mouse, guinea pig, rabbit, sheep, pig, bovine,horse, cat, dog, monkey, and so forth).

[0486] Dose levels of the above compound or a salt thereof may varydepending upon the target disease, the patient to be treated,administration route, and so on. When the compound is administeredorally, generally the compound is administered to adult patients withhypothyroidism (60 kg in body weight) at a dose of about 0.1-100 mg/day,preferably about 1.0-50 mg/day, more preferably about 1.0-20 mg/day.With respect to parenteral administration, when the compound isadministered to adult patients with hypothyroidism (60 kg in bodyweight), for example, in the form of an injection, it is convenient tointravenously inject the compound at a dose of about 0.01-30 mg/day,preferably about 0.1-20 mg/day, and more preferably about 0.1-10 mg/day,though the dose per administration may vary depending on the patient tobe treated, the target disease, and so forth. For other animals,corresponding doses may be administered after appropriate conversion ofthe above-mentioned values per 60 kg.

[0487] (10b) Method of Screening for Compounds that Promote or Inhibitthe Activity of the Promoter for the DNA of the Invention

[0488] The present invention provides a method of screening forcompounds, or salts thereof, that promote or inhibit the activity of thepromoter for the DNA of the invention, which is characterized byadministering a test compound to a non-human mammal deficient inexpression of the DNA of the invention and detecting the expression of areporter gene.

[0489] In the above screening method, there is used a non-human mammaldeficient in expression of the DNA of the invention wherein the DNA ofthe invention is inactivated as a result of introduction of a reportergene, and this reporter gene is capable of being expressed under thecontrol of the promoter for the DNA of the invention.

[0490] As the test compound, the compounds as enumerated above may beused.

[0491] As the reporter gene, the genes as enumerated above may be used.Among all, β-galactosidase gene (lacZ), soluble alkali phosphatase geneor luciferase gene may be preferably used.

[0492] In the non-human mammal deficient in expression of the DNA of theinvention wherein the DNA of the invention is replaced with a reportergene, since the reporter gene is present under the control of thepromoter for the DNA of the invention, the promoter activity can bedetected by tracing the expression of the substance encoded by thereporter gene.

[0493] For example, when a part of the DNA region encoding the RFRP-3 ofthe invention is replaced with E. coli-derived β-galactosidase gene(lacZ), β-galactosidase is expressed instead of the RFRP-3 of theinvention in those tissues where originally the RFRP-3 of the inventionhas been expressed. Thus, by staining with a substrate forβ-galactosidase such as5-bromo-4-chloro-3-indolyl-β-D-galactopyrasosidase (X-gal), it ispossible to observe the state of in vivo expression of the RFRP-3 of theinvention in the mammal simply. Specifically, mice deficient in theRFRP-3 of the invention or tissue sections thereof may be fixed inglutaraldehyde or the like, washed with phosphate-buffered physiologicalsaline (PBS), and treated with a staining solution containing X-gal atroom temperature or around 37° C. for about 30 min to 1 hr.Subsequently, the tissue samples are washed with 1 mM EDTA/PBS solutionto terminate the β-galactosidase reaction, followed by observation ofthe resultant color development. Alternatively, mRNA encoding lacZ maybe detected according to conventional methods.

[0494] The compounds or salts thereof obtainable by the above-describedscreening are compounds that are selected from the above-mentioned testcompounds and yet promote or inhibit the promoter activity for the DNAof the invention.

[0495] The compound obtained by the above screening may be in a saltform. As salts of the compound, salts formed with physiologicallyacceptable acids (e.g. organic or inorganic acids) or bases (e.g. alkalimetals) may be used. Especially preferable are physiologicallyacceptable acid addition salts. Examples of such salts include saltsformed with inorganic acids (e.g. hydrochloric acid, phosphoric acid,hydrobromic acid or sulfuric acid) and salts formed with organic acids(e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleicacid, succinic acid, tartaric acid, citric acid, malic acid, oxalicacid, benzoic acid, methanesulfonic acid or benzenesulfonic acid).

[0496] Since compounds, or salts thereof, that promote the promoteractivity for the DNA of the invention can promote the expression of theRFRP-3 of the invention and thereby promote the function thereof, theymay be used as prophylactic and/or therapeutic agents for variousdiseases associated with prolactin secretion insufficiency, and useful,based on their prolactin secretion-promoting effect, as prophylacticand/or therapeutic agents for various diseases associated with prolactinsecretion such as hypoovarianism, seminal vesicle hypoplasia,osteoporosis, menopausal syndrome, hypogalactia, hypothyroidism, renalinsufficiency, and so forth or paralgesia.

[0497] Further, since those compounds or salts thereof have aphrodisiaceffect (pheromone-like effect) based on their prolactinsecretion-promoting effect, they are also useful as an aphrodisiacagent.

[0498] Since compounds, or salts thereof, that inhibit the promoteractivity for the DNA of the invention can inhibit the expression of theRFRP-3 of the invention and thereby inhibit the function thereof, theymay be used as prophylactic and/or therapeutic agents for variousdiseases associated with excessive prolactin secretion, and useful,based on their prolactin secretion-inhibiting effect, as prophylacticand/or therapeutic agents for various diseases associated with prolactinsecretion such as hyperprolactinemia, pituitary adenoma, diencephalictumor, menstrual disorder, stresses, autoimmune diseases, prolactinoma,sterility, impotence, amenorrhea, galactorrhea, acromegaly,Chiari-Frommel syndrome, Argonzdel Castillo syndrome, Forbes-Albrightsyndrome, breast cancer lymphoma or Sheehan's syndrome or spermatogenicdisorder, and so forth.

[0499] Further, those compounds or salts thereof are also useful as acontraceptive based on their prolactin secretion-inhibiting effect.

[0500] Further, those compounds or salts thereof are also useful as ananalgesic agent.

[0501] Additionally, those compounds or salts thereof obtainable of theabove screening method are also useful as a medicine for testingprolactin secretion function or a veterinary medicine such as apromoting agent for milk secretion in mammals such as bovine, goat, pig,and so forth. Besides, it is expected to apply those compounds or saltsthereof to a useful substance production system where the usefulsubstance is allowed to be produced in mammal bodies and then secretedinto their milk.

[0502] Further, since those compounds or salts thereof have placentalfunction-regulating effect, they are also useful as a prophylacticand/or therapeutic agent for choriocarcinoma, hydatidiform mole,invasive mole, miscarriage, fetal hypotrophy, glucose metabolismdisorder, lipidosis or induction of delivery.

[0503] Further, compounds derived from those compounds obtained from theabove screening may also be used in the same manner.

[0504] Medicines comprising the compound or, a salt thereof, obtained bythe above screening may be prepared in the same manner as described formedicines comprising the RFRP-3 of the invention or a salt thereof.

[0505] Since the thus obtained preparations are safe and of lowtoxicity, they may be administered to, for example, human or othermammals (such as rat, mouse, guinea pig, rabbit, sheep, pig, bovine,horse, cat, dog, monkey, and so forth).

[0506] Dose levels of the above compound or a salt thereof may varydepending upon the target disease, the patient to be treated,administration route, and so on. When a compound that promotes thepromoter activity for the DNA of the invention is administered orally,generally the compound is administered to adult patients withhypothyroidism (60 kg in body weight) at a dose of about 0.1-100 mg/day,preferably about 1.0-50 mg/day, more preferably about 1.0-20 mg/day.With respect to parenteral administration, when a compound that promotesthe promoter activity for the DNA of the invention is administered toadult patients with hypothyroidism (60 kg in body weight), for example,in the form of an injection, it is convenient to intravenously injectthe compound at a dose of about 0.01-30 mg/day, preferably about 0.1-20mg/day, and more preferably about 0.1-10 mg/day, though the dose peradministration may vary depending on the patient to be treated, thetarget disease, and so forth. For other animals, corresponding doses maybe administered after appropriate conversion of the above-mentionedvalues per 60 kg.

[0507] On the other hand, when a compound that inhibits the promoteractivity for the DNA of the invention is administered orally, generallythe compound is administered to adult patients with hyperprolactinemia(60 kg in body weight) at a dose of about 0.1-100 mg/day, preferablyabout 1.0-50 mg/day, more preferably about 1.0-20 mg/day. With respectto parenteral administration, when a compound that inhibits the promoteractivity for the DNA of the invention is administered to adult patientswith hyperprolactinemia (60 kg in body weight) in the form of aninjection, it is convenient to intravenously inject the compound at adose of about 0.01-30 mg/day, preferably about 0.1-20 mg/day, and morepreferably about 0.1-10 mg/day, though the dose per administration mayvary depending on the patient to be treated, the target disease, and soforth. For other animals, corresponding doses may be administered afterappropriate conversion of the above-mentioned values per 60 kg.

[0508] Thus, the non-human mammal deficient in expression of the DNA ofthe invention is extremely useful in screening for compounds, or saltsthereof, that promote or inhibit the promoter activity for the DNA ofthe invention, and may contribute greatly to the elucidation of causesof various diseases resulted from deficiency in expression of the DNA ofthe invention, or to the development of prophylactic and/or therapeuticagents for such diseases.

[0509] When the so-called transgenic animal is created by using a DNAcomprising the promoter region of the RFRP-3 of the invention, ligatinggenes encoding various proteins downstream of the promoter region, andinjecting the thus prepared construct into egg cells of an animal, it ispossible to allow the animal to synthesize its RFRP-3 specifically,which will enable the examination of the in vivo function of the RRRP-3.Further, if a cell line which expresses an appropriate reporter geneligated to the above promoter region has been established, such a cellline may be used as a search system for those low molecular weightcompounds that specifically promote or inhibit in vivo productionability for the RFRP-3 of the invention per se.

[0510] In the specification and drawings of the present application, theabbreviations used for bases (nucleotides), amino acids and so forth arethose recommended by the IUPAC-IUB Commission on BiochemicalNomenclature or those conventionally used in the art. Examples of suchabbreviations are given below. Amino acids that may have optical isomersare intended to represent their L-isomer unless otherwise specified.

[0511] DNA: Deoxyribonucleic acid

[0512] cDNA: Complementary deoxyribonucleic acid

[0513] A: Adenine

[0514] T: Thymine

[0515] G: Guanine

[0516] C: Cytosine

[0517] I: Inosine

[0518] R: Adenine (A) or Guanine (G)

[0519] Y: Thymine M or Cytosine (C)

[0520] M: Adenine (A) or Cytosine (C)

[0521] K: Guanine (G) or Thymine MT)

[0522] S: Guanine (G) or Cytosine (C)

[0523] W: Adenine (A) or Thymine (I)

[0524] B: Guanine (G), Guanine (G) or Thymine Cr)

[0525] D: Adenine (A), Guanine (G) or Thymine Cr)

[0526] V: Adenine (A), Guanine (G) or Cytosine (C)

[0527] N: Adenine (A), Guanine (G), Cytosine (C) or Thymine (I), orunknown or other nucleotide

[0528] RNA: Ribonucleic acid

[0529] mRNA: Messenger ribonucleic acid

[0530] dATP: Deoxyadenosine triphosphate

[0531] dTTP: Deoxythymidine triphosphate

[0532] dGTP: Deoxyguanosine triphosphate

[0533] dCTP: Deoxycytidine triphosphate

[0534] ATP: Adenosine triphosphate

[0535] EDTA: Ethylenediaminetetracetic acid

[0536] SDS: Sodium dodecyl sulfate

[0537] BHA: Banzhydrylamine

[0538] pMBHA: p-Methylbenzhydrylamine

[0539] Tos: p-Toluene sulfonyl

[0540] Bzl: Benzyl

[0541] Bom: Benzyloxymethyl

[0542] Boc: t-Butyloxycarbonyl

[0543] DCM: Dichloromethane

[0544] HOBt: 1-Hydroxybenzotriazole

[0545] DCC: N,N′-Dicyclohexylcarbodiimide

[0546] TFA: Trifluoroacetate

[0547] DIEA: Diisopropylethylamine

[0548] Gly: Glycine

[0549] Ala or A: Alanine

[0550] Val or V: Valine

[0551] Leu or L: Leucine

[0552] Ile or I: Isoleucine

[0553] Ser or S: Serine

[0554] Thr or T: Threonine

[0555] Cys or C: Cysteine

[0556] Met or M: Methionine

[0557] Glu or E: Glutamic acid

[0558] Asp or D: Aspartic acid

[0559] Lys or K: Lysine

[0560] Arg or R: Arginine

[0561] His or H: Histidine

[0562] Phe or F: Phenylalanine

[0563] Tyr or Y: Tyrosine

[0564] Trp or W: Tryptophan

[0565] Pro or P: Proline

[0566] Asn or N: Asparagine

[0567] Gln or Q: Glutamine

[0568] pGlu: Pyroglutamic acid

[0569] BHA: Banzhydrylanine

[0570] pMBHA: p-Methylbenzhydrylamine

[0571] Tos: p-Toluene sulfonyl

[0572] Bzl: Benzyl

[0573] OcHex: Cyclohexyl ester

[0574] Boc: t-Butyloxycarbonyl

[0575] DCM: Dichoromethane

[0576] HOBt: 1-Hydroxybenztriazole

[0577] DCC: N,N′-Dicyclohexylcarbodiimide

[0578] TFA: Trifluoroacetate

[0579] DIEA: Diisopropylethylamine

[0580] The SEQ ID NOS of the SEQUENCE LISTING of the presentspecification represent the sequences as indicated below.

[0581] [SEQ ID NO: 1]

[0582] This shows the amino acid sequence of the polypeptide of theinvention (human type) obtained in Reference Example 1 described later.

[0583] [SEQ ID NO: 2]

[0584] This shows the nucleotide sequence of a DNA encoding thepolypeptide of the invention comprising the amino acid sequencerepresented by SEQ ID NO: 1.

[0585] [SEQ ID NO: 3]

[0586] This shows the nucleotide sequence of primer F5 used in theReference Example 1 described later.

[0587] [SEQ ID NO: 4]

[0588] This shows the nucleotide sequence of primer F6 used in theReference Example 1 described later.

[0589] [SEQ ID NO: 5]

[0590] This shows the nucleotide sequence of primer F1 used in theReference Example 1 described later. [SEQ ID NO: 6]

[0591] This shows the nucleotide sequence of primer R5 used in theReference Example 1 described later.

[0592] [SEQ ID NO: 7]

[0593] This shows the nucleotide sequence of primer hR1 used in theReference Example 3 described later.

[0594] [SEQ ID NO: 8]

[0595] This shows the amino acid sequence of the polypeptide of theinvention (human type) obtained in Reference Example 3 described later.

[0596] [SEQ ID NO: 9]

[0597] This shows the nucleotide sequence of a DNA encoding thepolypeptide of the invention comprising the amino acid sequencerepresented by SEQ ID NO: 8.

[0598] [SEQ ID NO: 10]

[0599] This shows the nucleotide sequence of primer bF6 used in theReference Example 4 described later.

[0600] [SEQ ID NO: 11]

[0601] This shows the nucleotide sequence of primer bF7 used in theReference Example 4 described later.

[0602] [SEQ ID NO: 12]

[0603] This shows the nucleotide sequence of primer bR6 used in theReference Example 4 described later.

[0604] [SEQ ID NO: 13]

[0605] This shows the nucleotide sequence of primer bR7 used in theReference Example 4 described later.

[0606] [SEQ ID NO: 14]

[0607] This shows the amino acid sequence of the polypeptide of theinvention (bovine type) obtained in the Reference Example 4 describedlater.

[0608] [SEQ ID NO: 15]

[0609] This shows the nucleotide sequence of a DNA encoding thepolypeptide of the invention comprising the amino acid sequencerepresented by SEQ ID NO: 14.

[0610] [SEQ ID NO: 16]

[0611] This shows the nucleotide sequence of primer rLPR1 used in theReference Example 5 described later.

[0612] [SEQ ID NO: 17]

[0613] This shows the nucleotide sequence of primer rLPF1 used in theReference Example 5 described later. [SEQ ID NO: 18]

[0614] This shows the amino acid sequence of the polypeptide of theinvention (rat type) obtained in the Reference Example 5 described later(before re-cloning).

[0615] [SEQ ID NO: 19]

[0616] This shows the nucleotide sequence of a DNA encoding thepolypeptide of the invention comprising the amino acid sequencerepresented by SEQ ID NO: 18.

[0617] [SEQ ID NO: 20]

[0618] This shows a nucleotide sequence encoding RFGK sequence.

[0619] [SEQ ID NO: 21]

[0620] This shows a nucleotide sequence encoding RFGR sequence.

[0621] [SEQ ID NO: 22]

[0622] This shows a nucleotide sequence encoding RSGK sequence.

[0623] [SEQ ID NO: 23]

[0624] This shows a nucleotide sequence encoding RSGR sequence.

[0625] [SEQ ID NO: 24]

[0626] This shows a nucleotide sequence encoding RLGK sequence.

[0627] [SEQ ID NO: 25]

[0628] This shows a nucleotide sequence encoding RLGR sequence.

[0629] [SEQ ID NO: 26]

[0630] This shows the nucleotide sequence of primer FF2 used in theReference Example 6 described later.

[0631] [SEQ ID NO: 27]

[0632] This shows the nucleotide sequence of primer rR4 used in theReference Example 6 described later.

[0633] [SEQ ID NO: 28]

[0634] This shows the nucleotide sequence of primer mF1 used in theReference Example 6 described later.

[0635] [SEQ ID NO: 29]

[0636] This shows the nucleotide sequence of primer mF3 used in theReference Example 6 described later.

[0637] [SEQ ID NO: 30]

[0638] This shows the nucleotide sequence of primer mRR1 used in theReference Example 6 described later.

[0639] [SEQ ID NO: 31]

[0640] This shows the nucleotide sequence of primer moF used in theReference Example 6 described later.

[0641] [SEQ ID NO: 32]

[0642] This shows the nucleotide sequence of primer moR used in theReference Example 6 described later.

[0643] [SEQ ID NO: 33]

[0644] This shows the amino acid sequence of the polypeptide of theinvention (mouse type) obtained in the Reference Example 6 describedlater.

[0645] [SEQ ID NO: 34]

[0646] This shows the nucleotide sequence of a DNA encoding thepolypeptide of the invention comprising the amino acid sequencerepresented by SEQ ID NO: 33.

[0647] [SEQ ID NO: 35]

[0648] This shows the nucleotide sequence of primer 1 used for cloningthe cDNA encoding the novel G protein-coupled receptor protein rOT7T022Lderived from the peripheral area of rat brainstem obtained in ReferenceExample 7 described later.

[0649] [SEQ ID NO: 36]

[0650] This shows the nucleotide sequence of primer 2 used for cloningthe cDNA encoding the novel G protein-coupled receptor protein rOT7T022Lderived from the peripheral area of rat brainstem obtained in ReferenceExample 7 described later.

[0651] [SEQ ID NO: 37]

[0652] This shows the amino acid sequence of the novel G protein-coupledreceptor protein rOT7T022L derived from the peripheral area of ratbrainstem obtained in Reference Example 7 described later.

[0653] [SEQ ID NO: 38]

[0654] This shows the nucleotide sequence of the cDNA encoding the novelG protein-coupled receptor protein rOT7T022L derived from the peripheralarea of rat brainstem obtained in Reference Example 7 described later.

[0655] [SEQ ID NO: 39]

[0656] This shows the amino acid sequence of the peptide obtained in (3)in Reference Example 7 described later.

[0657] [SEQ ID NO: 40]

[0658] This shows the amino acid sequence of the peptide obtained in (4)in Reference Example 7 described later.

[0659] [SEQ ID NO: 41]

[0660] This shows the amino acid sequence of the peptide obtained in (5)in Reference Example 7 described later.

[0661] [SEQ ID NO: 42]

[0662] This shows a nucleotide sequence encoding a peptide comprising anamino acid sequence which is from position 81 (Met) to position 92 (Phe)of the amino acid sequence represented by SEQ ID NO: 1.

[0663] [SEQ ID NO: 43]

[0664] This shows a nucleotide sequence encoding a peptide comprising anamino acid sequence which is from position 101 (Ser) to position 112(Ser) of the amino acid sequence represented by SEQ ID NO: 1.

[0665] [SEQ ID NO: 44]

[0666] This shows a nucleotide sequence encoding a peptide comprising anamino acid sequence which is from position 124 (Val) to position 131(Phe) of the amino acid sequence represented by SEQ ID NO: 1.

[0667] [SEQ ID NO: 45]

[0668] This shows a nucleotide sequence encoding a peptide comprising anamino acid sequence which is from position 1 (Met) to position 92 (Phe)of the amino acid sequence represented by SEQ ID NO: 1.

[0669] [SEQ ID NO: 46]

[0670] This shows a nucleotide sequence encoding a peptide comprising anamino acid sequence which is from position 1 (Met) to position 112 (Ser)of the amino acid sequence represented by SEQ ID NO: 1.

[0671] [SEQ ID NO: 47]

[0672] This shows a nucleotide sequence encoding a peptide comprising anamino acid sequence which is from position 1 (Met) to position 131 (Phe)of the amino acid sequence represented by SEQ ID NO: 1.

[0673] [SEQ ID NO: 48]

[0674] This shows the nucleotide sequence of primer ratF2 used inReference Example 5.

[0675] [SEQ ID NO: 49]

[0676] This shows the nucleotide sequence of primer ratR used inReference Example 5.

[0677] [SEQ ID NO: 50]

[0678] This shows the amino acid sequence of the polypeptide of theinvention (rat type) obtained in the Reference Example 5 described later(after re-cloning).

[0679] [SEQ ID NO: 51]

[0680] This shows the nucleotide sequence of a DNA encoding thepolypeptide of the invention comprising the amino acid sequencerepresented by SEQ ID NO: 50.

[0681] [SEQ ID NO: 52]

[0682] This shows the nucleotide sequence of primer bFF used inReference Example 9.

[0683] [SEQ ID NO: 53]

[0684] This shows the nucleotide sequence of primer bFR used inReference Example 9.

[0685] [SEQ ID NO: 54]

[0686] This shows the amino acid sequence encoding the protein(polypeptide) designated hOT7T022 obtained in Reference Example 11.

[0687] [SEQ ID NO: 55]

[0688] This shows the nucleotide sequence of a DNA encoding the protein(polypeptide) designated hOT7T022 comprising the amino acid sequencerepresented by SEQ ID NO: 54.

[0689] [SEQ ID NO: 56]

[0690] This shows the nucleotide sequence of a DNA encoding the protein(polypeptide) designated hOT7T022 comprising the amino acid sequencerepresented by SEQ ID NO: 54.

[0691] [SEQ ID NO: 57]

[0692] This shows the nucleotide sequence of primer 1 used in ReferenceExample 11.

[0693] [SEQ ID NO: 58]

[0694] This shows the nucleotide sequence of primer 2 used in ReferenceExample 11.

[0695] [SEQ ID NO: 59]

[0696] This shows the nucleotide sequence of primer #1 used in ExampleA4.

[0697] [SEQ ID NO: 60]

[0698] This shows the nucleotide sequence of primer #2 used in ExampleA4.

[0699] [SEQ ID NO: 61]

[0700] This shows the nucleotide sequence of primer #3 used in ExampleA4.

[0701] [SEQ ID NO: 62]

[0702] This shows the nucleotide sequence of primer #4 used in ExampleA4.

[0703] [SEQ ID NO: 63]

[0704] This shows the amino acid sequence of a peptide comprising anamino acid sequence which is from position 104 (Ala) to position 131(Phe) of the amino acid sequence represented by SEQ ID NO: 1.

[0705] [SEQ ID NO: 64]

[0706] This shows the nucleotide sequence of a DNA encoding a peptidecomprising an amino acid sequence which is from position 104 (Ala) toposition 131 (Phe) of the amino acid sequence represented by SEQ ID NO:1.

[0707] [SEQ ID NO: 65]

[0708] This shows the amino acid sequence of a peptide comprising anamino acid sequence which is from position 101 (Ser) to position 131(Phe) of the amino acid sequence represented by SEQ ID NO: 1.

[0709] [SEQ ID NO: 66]

[0710] This shows the nucleotide sequence of a DNA encoding a peptidecomprising an amino acid sequence which is from position 101 (Ser) toposition 131 (Phe) of the amino acid sequence represented by SEQ ID NO:1.

[0711] [SEQ ID NO: 67]

[0712] This shows the amino acid sequence of a peptide comprising anamino acid sequence which is from position 104(Ala) to position 131(Phe) of the amino acid sequence represented by SEQ ID NO: 14.

[0713] [SEQ ID NO: 68]

[0714] This shows the nucleotide sequence of a DNA encoding a peptidecomprising an amino acid sequence which is from position 104 (Ala) toposition 131 (Phe) of the amino acid sequence represented by SEQ ID NO:14.

[0715] [SEQ ID NO: 69]

[0716] This shows the amino acid sequence of peptide RFRP-3(5)comprising an amino acid sequence which is from position 127 (Leu) toposition 131 (Phe) of the amino acid sequence represented by SEQ ID NO:1 or SEQ ID NO: 12.

[0717] [SEQ ID NO: 70]

[0718] This shows the amino acid sequence of peptide RFRP-3(6)comprising an amino acid sequence which is from position 126 (Asn) toposition 131 (Phe) of the amino acid sequence represented by SEQ ID NO:1 or SEQ ID NO: 12.

[0719] [SEQ ID NO: 71]

[0720] This shows the amino acid sequence of peptide RFRP-3(7)comprising an amino acid sequence which is from position 125 (Pro) toposition 131 The) of the amino acid sequence represented by SEQ ID NO: 1or SEQ ID NO: 12.

[0721] [SEQ ID NO: 72]

[0722] This shows the amino acid sequence of peptide RFRP-3(8)comprising an amino acid sequence which is from position 124 (Val) toposition 131 (Phe) of the amino acid sequence represented by SEQ ID NO:1 or SEQ ID NO: 12.

[0723] Transformant Escherichia coli JM109/phRF1 obtained in ReferenceExample 2 described later has been deposited with the InternationalPatent Organism Depository, National Institute of Advanced IndustrialScience and Technology (former designation: National Institute ofBioscience and Human Technology, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry (NJBH)) locatedat Central 6, 1-1 Higashi 1-chome, Tsukuba City, Ibaraki Pref., Japan(zip code No.: 305-8566) since Apr. 14, 1999 under the Accession No.FERM BP-6702, and with the Institute for Fermentation, Osaka (IFO) sinceMar. 5, 1999 under the Accession No. IFO 16265.

[0724] Transformant Escherichia coli DH10B/pAK-rOT022L obtained inReference Example 7 described later has been deposited with theInternational Patent Organism Depository, National Institute of AdvancedIndustrial Science and Technology (former designation: NIBH) since Nov.2, 1998 under the Accession No. FERM BP-6558, and with the Institute forFermentation, Osaka (IFO) since Oct. 16, 1998 under the Accession No.IFO 16211.

[0725] Transformant Escherichia coli JM109/pbRF2 obtained in ReferenceExample 9 described later has been deposited with the InternationalPatent Organism Depository, National Institute of Advanced IndustrialScience and Technology (former designation: NIBH) since Aug. 2, 1999under the Accession No. FERM BP-6811, and with the Institute forFermentation, Osaka (IFO) since Jun. 18, 1999 under the Accession No.IFO 16288.

[0726] Transformant Escherichia coli JM109/phRF2 obtained in ReferenceExample 8 described later has been deposited with the InternationalPatent Organism Depository, National Institute of Advanced IndustrialScience and Technology (former designation: NIBH) since Aug. 2, 1999under the Accession No. FERM BP-6812, and with the Institute forFermentation, Osaka (IFO) since Jun. 18, 1999 under the Accession No.IFO 16289.

[0727] Transformant Escherichia coli JM109/pmLP4 obtained in ReferenceExample 6 described later has been deposited with the InternationalPatent Organism Depository, National Institute of Advanced IndustrialScience and Technology (former designation: NIBH) since Aug. 2, 1999under the Accession No. FERM BP-6813, and with the Institute forFermentation, Osaka (IFO) since Jun. 18, 1999 under the Accession No.IFO 16290.

[0728] Transformant Escherichia coli JM109/prLOL6 obtained in ReferenceExample 5 described later has been deposited with the InternationalPatent Organism Depository, National Institute of Advanced IndustrialScience and Technology (former designation: NIBH) since Aug. 2, 1999under the Accession No. FERM BP-6814, and with the Institute forFermentation, Osaka (IFO) since Jun. 18, 1999 under the Accession No.IFO 16291.

[0729] Transformant Escherichia coli DH5α/pCR2.1-hOT022T obtained inReference Example 11 described later has been deposited with theInternational Patent Organism Depository, National Institute of AdvancedIndustrial Science and Technology (former designation: NIBH) since Nov.8, 1999 under the Accession No. FERM BP-6930, and with the Institute forFermentation, Osaka (IFO) since Oct. 27, 1999 under the Accession No.IFO 16330.

[0730] Transformant Escherichia coli DH5α/pCR2.1-hOT022G obtained inReference Example 11 described later has been deposited with theInternational Patent Organism Depository, National Institute of AdvancedIndustrial Science and Technology (former designation: NIBH) since Nov.8, 1999 under the Accession No. FERM BP-6931, and with the Institute forFermentation, Osaka (IFO) since Oct. 27, 1999 under the Accession No.IFO 16331.

[0731] Transformant Escherichia coli MM294 (DE3)/pTFCRFRP-1 obtained inExample A5 described later has been deposited with the InternationalPatent Organism Depository, National Institute of Advanced IndustrialScience and Technology (former designation: NIBH) since Sep. 28, 2000under the Accession No. FERM BP-7313, and with the Institute forFermentation, Osaka (IFO) since Sep. 19, 2000 under the Accession No.IFO 16476.

[0732] Hybridoma IF3 obtained in Reference Example 12 described laterwhich produces anti-rat RFRP-1 monoclonal antibody has been depositedwith the International Patent Organism Depository, National Institute ofAdvanced Industrial Science and Technology (former designation: NIBH)since Feb. 21, 2001 under the Accession No. FERM BP-7463, and with theInstitute for Fermentation, Osaka (IFO) since Jan. 16, 2001 under theAccession No. IFO 50527.

EXAMPLES

[0733] Hereinbelow, the present invention will be described in moredetail with reference to the following Reference Examples and Examples.However, the present invention is not limited to these ReferenceExamples and Examples. The genetic engineering procedures usingEscherichia coli were in accordance with those procedures described inthe book “Molecular Cloning”.

Reference Example 1 Synthesis of cDNA from Human Fetal Brain Poly(A)⁺RNA Fraction and Amplification of a cDNA Encoding Physiologically ActivePeptide by RT-PCR

[0734] Oligo dT primer (Gibco BRL) was added to 1 μg of a human fetalbrain poly(A)⁺ RNA fraction purchased from Clontech. Using a reversetranscriptase from Moloney murine leukemia virus (Gibco BRL) and itsaccompanied buffer, cDNA was synthesized. The reaction product wasextracted with phenol:chloroform (1:1), precipitated with phenol andthen dissolved in 30 μl of TE. Using 1 μl of the thus prepared cDNA as atemplate, PCR amplification was carried out with following two primers(F5 and F6). F5: 5′-GGGCTGCACATAGAGACTTAATTTTAG-3′ (SEQ ID NO: 3) F6:5′-CTAGACCACCTCTATATAACTGCCCAT-3′ (SEQ ID NO: 4)

[0735] The composition of the reaction solution was as follows: 20 pMeach of synthetic DNA primers (F5 and F6), 0.25 mM dNTPs, 0.5 μl of ExTaq DNA polymerase and 5 μl of its accompanied buffer. The total volumeof the reaction solution was 50 μl. For amplification, 40 cycles of 98°C. for 10 sec, 63° C. for 20 sec and 72° C. for 40 sec were performed ina thermal cycler (Perkin Elmer).

[0736] Further, using 1 μl of the resultant PCR product as a template,amplification was performed by nested PCR using the following twoprimers (F1 and R5). F1: 5′-GCACATAGAGACTTAATTTTAGATTTAGAC-3′ (SEQ IDNO: 5) R5: 5′-CATGCACTTTGACTGGTTTCCAGGTAT-3′ (SEQ ID NO: 6)

[0737] The composition of the reaction solution was as follows: 20 pMeach of synthetic DNA primers (F1 and R5), 0.25 mM dNTPs, 0.5 μl of ExTaq DNA polymerase and 5 μl of its accompanied buffer. The total volumeof the reacton solution was 50 μl. For amplification, 40 cycles of 98°C. for 10 sec, 60° C. for 20 sec and 72° C. for 40 sec were performed ina thermal cycler (Perkin Elmer). The amplified product was confirmed by1.2% agarose gel electrophoresis and ethidium bromide staining.

Reference Example 2 Subcloning of the PCR Product into Plasmid Vectorand Selection of Candidate Clones for Novel Physiologically ActivePeptide by Analysis of the Nucleotide Sequence of the Inserted cDNA

[0738] The reaction product obtained by PCR in Reference Example 1 wasseparated using 1.2% agarose gel. Amplification of a DNA fragment of theexpected size was confirmed. Then, the DNA was recovered using QuiagenPCR Purification kit (Quiagen). According to the protocol attached to aTA cloning kit (Invitrogen), the recovered DNA was subcloned intoplasmid vector pCR™2.1. This plasmid vector was introduced into E. coliJM109 competent cells (Takara Shuzo) to prepare transformants. From theresultant transformants, those clones carrying the inserted cDNAfragment were selected in LB agar medium containing ampicillin, IPTG andX-gal. Only those clones presenting a white color were isolated with asterilized tooth pick to thereby obtain transformant Escherichia coliJM109/phRF1.

[0739] Each of these clones was cultured overnight inampicillin-containing LB medium and then plasmid DNA was preparedtherefrom using an automated plasmid extraction apparatus (Kurabo). Apart of the thus prepared DNA was digested with EcoRI, followed byconfirmation of the size of the inserted cDNA fragment. A part of theremaining DNA was treated with RNase, extracted with phenol:chloroform,and then precipitated with ethanol for concentration. Reactions fordetermining the nucleotide sequence were performed with Dye DeoxyTerminator Cycle Sequencing Kit (ABI), followed by reading with afluorescence-based automated sequencer. The resultant information aboutthe nucleotide sequence was analyzed using DNASIS (Hitachi SystemEngineering). The thus determined nucleotide sequence is shown in FIG.1.

[0740] As a result of homology search and sequence analysis based onFIG. 1, it was found that the cDNA fragment inserted into the plasmidcarried by transformant E. coli JM109/phRF1 encodes a novel,physiologically active peptide.

Reference Example 3 Acquisition of a Splicing Variant of the cDNAEncoding Physiologically Active Peptide from Human Fetal Brain cDNA

[0741] Using 1 ml of the human fetal brain cDNA prepared in ReferenceExample 1 as a template, PCR amplification was performed with thefollowing two primers (F5 and hR1). F5:5′-GGGCTGCACATAGAGACTTAATTTTAG-3′ (SEQ ID NO: 3) hR1:5′-CAGCTTTAGGGACAGGCTCCAGGTTTC-3′ (SEQ ID NO: 7)

[0742] The composition of the reaction solution was as follows: 20 pMeach of synthetic DNA primers (F5 and hR1), 0.25 mM dNTPs, 0.5 ml of ExTaq DNA polymerase and its accompanied buffer. The total volume of thereacton solution was 50 ml. For amplification, 40 cycles of 98° C. for10 sec, 65° C. for 20 sec and 72° C. for 20 sec were performed in athermal cycler (Perkin Elmer). The amplified product was confirmed by1.2% agarose gel electrophoresis and ethidium bromide staining. Afterthe confirmation of amplification of the PCR product, the reactionproduct was purified with QIA Quick PCR Purification Kit (Quiagen) andthen sequenced. Reactions for determining the nucleotide sequence wereperformed with Big Dye Deoxy Terminator Cycle Sequencing Kit (ABI),followed by reading with a fluorescence-based automated sequencer(ABI377). The resultant information about the nucleotide sequence wasanalyzed using DNASIS (Hitachi System Engineering). As a result, a cDNAwhose 3′-terminal region was different from that of the cDNA obtained inReference Example 2 was obtained. It was found that this cDNA was asplicing variant of the cDNA obtained in Reference Example 2. Thenucleotide sequence determined (SEQ ID NO: 9) and the amino acidsequence deduced therefrom (SEQ ID NO: 8) are shown in FIG. 3.

Reference Example 4 Acquisition of a cDNA Encoding PhysiologicallyActive Peptide from Bovine Hypothalamus Poly(A)⁺ RNA

[0743] A cDNA encoding a bovine-type physiologically active peptide wasobtained from bovine hypothalamus poly(A)⁺ RNA using Marathon cDNAAmplification Kit (Clontech). Briefly, bovine hypothalamus cDNA wasprepared according to the manual attached to the kit. Using this cDNA asa template, PCR amplifications were performed with the following foursynthetic primers (bF6, bF7, bR6 and bR7) in combination with twoaccompanied primers AP1 and AP2 to the kit. bF6:5′-GCCTAGAGGAGATCTAGGCTGGGAGGA-3′ (SEQ ID NO: 10) bF7:5′-GGGAGGAACATGGAAGAAGAAAGGAGC-3′ (SEQ ID NO: 11) bR6:5′-GATGGTGAATGCATGGACTGCTGGAGC-3′ (SEQ ID NO: 12) bR7:5′-TTCCTCCCAAATCTCAGTGGCAGGTTG-3′ (SEQ ID NO: 13)

[0744] In order to amplify the 5′-terminal (N-terminal) region, thefirst PCR reaction was performed using two synthetic primers (bR6 andAP1). The reaction solution contained 20 pM each of the primers, 0.25 mMdNTPs, 0.5 ml of Klen Taq DNA Polymerase and the accompanied buffer tothe polymerase to make the total volume 25 ml. For amplification, 5cycles of 98° C. for 10 sec and 72° C. for 2 min; 5 cycles of 98° C. for10 sec and 70° C. for 2 min; and 25 cycles of 98° C. for 10 sec and 68°C. for 2.5 min were performed in a thermal cycler (Perkin Elmer).Subsequently, the resultant first PCR reaction solution was diluted10-fold. Using 1 ml of this dilution as a template, the second PCR wasperformed with primers bR7 and AP2. The reaction solution contained 20pM each of the primers, 0.25 mM dNTPs, 0.5 ml of Klen Taq DNA Polymeraseand the accompanied buffer to the polymerase to make the total volume 25ml. For amplification, 5 cycles of 98° C. for 10 sec and 72° C. for 2min; 5 cycles of 98° C. for 10 sec and 70° C. for 2 min; and 35 cyclesof 98° C. for 10 sec and 68° C. for 2.5 min were performed in a thermalcycler (Perkin Elmer).

[0745] In order to amplify the 3′-terminal (C-terminal) region, thefirst PCR reaction was performed using two synthetic primers (bF6 andAP1). The reaction solution contained 20 pM each of the primers, 0.25 mMdNTPs, 0.5 ml of Klen Taq DNA Polymerase and the accompanied buffer tothe polymerase to make the total volume 25 ml. For amplification, 5cycles of 98° C. for 10 sec and 72° C. for 2 min; 5 cycles of 98° C. for10 sec and 70° C. for 2 min; and 25 cycles of 98° C. for 10 sec and 68°C. for 2.5 min were performed in a thermal cycler (Perkin Elmer).Subsequently, the resultant first PCR reaction solution was diluted10-fold. Using 1 ml of this dilution as a template, the second PCR wasperformed with primers bF7 and AP2. The reaction solution contained 20pM each of the primers, 0.25 mM dNTPs, 0.5 ml of Klen Taq DNA Polymeraseand the accompanied buffer to the polymerase to make the total volume 25ml. For amplification, 5 cycles of 98° C. for 10 sec and 72° C. for 2min; 5 cycles of 98° C. for 10 sec and 70° C. for 2 min; and 35 cyclesof 98° C. for 10 sec and 68° C. for 2.5 min were performed in a thermalcycler (Perkin Elmer). Confirmation of the 5′-terminal and 3′-terminalamplified products were performed by 1.2% agarose gel electrophoresisand ethidium bromide staining. After the confirmation of amplificationof the PCR products, the reaction products were purified with QIA QuickPCR Purification Kit (Quiagen) and then sequenced. Reactions fordetermining the nucleotide sequences were performed with Big Dye DeoxyTerminator Cycle Sequencing Kit (ABI), followed by reading with afluorescence-based automated sequencer (ABI377).

[0746] The resultant information about the nucleotide sequence wasanalyzed using DNASIS (Hitachi System Engineering). The nucleotidesequence determined (SEQ ID NO: 15) and the amino acid sequence deducedtherefrom (SEQ ID NO: 14) are shown in FIG. 4.

Reference Example 5 Acquisition of a cDNA Encoding PhysiologicallyActive Peptide from Rat Brain Poly(A)⁺ RNA

[0747] A cDNA encoding a bovine-type physiologically active peptide wasobtained from rat brain poly(A)⁺ RNA using Marathon cDNA AmplificationKit (Clontech). Briefly, rat brain cDNA was prepared according to themanual attached to the kit. Using this cDNA as a template, PCRamplifications were performed with the following two synthetic primers:rLPR1: 5′-CCCTGGGGCTTCTTCTGTCTTCTATGT-3′ (SEQ ID NO: 16) rLPF1:5′-AGCGATTCATTTTATTGACTTTAGCA-3′ (SEQ ID NO: 17)

[0748] in combination with two accompanied primers AP1 and AP2 to thekit.

[0749] In order to amplify the 5′-terminal (1-terminal) region, thefirst PCR reaction was performed using primers rLPR1 and AP1. Thereaction solution contained 200 pM each of the primers, 0.1 mM each ofdNTPs, 0.25 ml of Klen Taq DNA Polymerase and the accompanied buffer tothe polymerase to make the total volume 25 ml. For amplification, 5cycles of 98° C. for 10 sec and 72° C. for 2 min; 5 cycles of 98° C. for10 sec and 70° C. for 2 min; and 25 cycles of 98° C. for 10 sec and 68°C. for 2.5 min were performed in a thermal cycler (Perkin Elmer).Subsequently, using the resultant reaction solution as a template, thesecond PCR was performed with the same primer set used in the first PCR.The composition of the reaction solution was the same as that for thefirst PCR. For amplification, 5 cycles of 98° C. for 10 sec and 72° C.for 2 min; 5 cycles of 98° C. for 10 sec and 70° C. for 2 min; and 38cycles of 98° C. for 10 sec (and 68° C. for 2.5 min) were performed in athermal cycler (Perkin Elmer).

[0750] In order to amplify the 3′-terminal (C-terminal) region, thefirst PCR reaction was performed using primers rLPF1 and AP1. Thecomposition of the reaction solution was the same as that for theamplification of the 5′-terminal (N-terminal region). For amplification,cycles of 98° C. for 10 sec and 72° C. for 2 min; 5 cycles of 98° C. for10 sec and 70° C. for 2 min; and 25 cycles of 98° C. for 10 sec, 65° C.for 20 sec and 72° C. for 2 min were performed in a thermal cycler(Perkin Elmer). Subsequently, using the resultant first PCR reactionsolution as a template, the second PCR was performed with primers rLPF1and AP2. The composition of the reaction solution was the same as thatfor the first PCR. For amplification, 5 cycles of 98° C. for 10 sec and72° C. for 2 min; 5 cycles of 98° C. for 10 sec and 70° C. for 2 min;and 38 cycles of 98° C. for 10 sec, 65° C. for 20 sec and 72° C. for 2min were performed in a thermal cycler (Perkin Elmer). Confirmation ofthe 5′-terminal and 3′-terminal amplified products were performed by1.2% agarose gel electrophoresis and ethidium bromide staining. Bands ofPCR product were purified with QIA Quick Gel Extraction Kit (Quiagen)and then sequenced. Determination of the nucleotide sequences wasperformed in the same manner as described in Reference Example 3. Thenucleotide sequence determined (SEQ ID NO: 19) and the amino acidsequence deduced therefrom (SEQ ID NO: 18) are shown in FIG. 5. Further,based on this nucleotide sequence, the following two primers weresynthesized near the initiation codon and the stop codon, respectively.ratF2: 5′-AATGGAAATTATTTCATCAAAGCGATT (SEQ ID NO: 48) CAT-3′ ratR:5′-CACCTATACTGACAGGAATGATGGCTC (SEQ ID NO: 49) TCC-3′

[0751] From rat hypothalamus poly(A)⁺ RNA, cDNA was synthesized using anAMV reverse transcriptase (Takara Shuzo) and random 9-mer (TakaraShuzo). Using this cDNA as a template, PCR reaction was performed for 33cycles of 98° C. for 10 sec and 68° C. for 40 sec. Subsequently, usingthe resultant reaction solution as a template, PCR reaction wasperformed for 38 cycles of 98° C. for 10 sec and 68° C. for 1 min, tothereby obtain a PCR product of approximately 690 bp. This product wasintroduced into cloning vector pCR2.1 TOPO according to the manual of TACloning Kit (Invitrogen), which was then introduced into E. coli JM109to thereby obtain transformant E. coli JM109/prLPL6. The nucleotidesequence was determined in the same manner as in Reference Example 3(SEQ ID NO: 51), and the amino acid sequence was deduced therefrom (SEQID NO: 50).

Reference Example 6 Acquisition of a cDNA Encoding Mouse-TypePhysiologically Active Peptide from Mouse Brain Poly(A)⁺ RNA by MarathonPCR and Confirmation of Its Sequence

[0752] In order to obtain a cDNA encoding a mouse-type physiologicallyactive peptide, first, 1 μg of mouse brain poly(A)⁺ RNA was reacted withSuperScriptII RNase H-reverse transcriptase (GIBCO BRL) in the presenceof 2.5 pmol of oligo d(T) primers (Takara Shuzo), 0.5 mM dNTPs and 10 mMDTT at 42° C. for 1 hr to synthesize cDNA. Using this cDNA as atemplate, PCR amplification was performed with the following primers:FF2: 5′-GACTTAATTTTAGATTTAGACAAAATGG (SEQ ID NO: 26) AA-3′ rR4:5′-TTCTCCCAAACCTTTGGGGCAGGTT-3′ (SEQ ID NO: 27)

[0753] and Klen Taq DNA polymerase (Clontech) for 39 cycles of 98° C.for 10 sec, 56° C. for 20 sec and 72° C. for 25 sec. Further, using thesame primer set, PCR reaction was performed for 25 cycles of 98° C. for10 sec, 60° C. for 20 sec and 72° C. for 25 sec. The resultant productwas detected by 2% agarose gel electrophoresis and ethidium bromidestaining. The band was purified with QIA Quick Gel Extraction Kit(Qiagen) and then sequenced in the same manner as in Reference Example3. In order to obtain the 5′-terminal and 3′-terminal sequences to theresultant cDNA fragment encoding a mouse-type physiologically activepeptide, cDNA was synthesized from 1 a g of mouse brain poly(A)⁺ RNAusing Marathon cDNA Amplification Kit (Clontech) in the same manner asin Reference Example 5 to thereby prepare a template. The followingthree primers: mF1: 5′-ACAGCAAAGAAGGTGACGGAAAATACTC-3′ (SEQ ID NO: 28)mF3: 5′-ATAGATGAGAAAAGAAGCCCCGCAGCAC-3′ (SEQ ID NO: 29) mR1:5′-GTGCTGCGGGGCTTCTTTTCTCATCTAT-3′ (SEQ ID NO: 30)

[0754] were synthesized and used in combination with the accompaniedprimer AP1 to the kit to perform PCR reactions.

[0755] In order to amplify the 5′-terminal (N-terminal) region, thefirst PCR reaction was performed using primers mR1 and AP1. For theamplification of the 3′-terminal (C-terminal) region, the first PCRreaction was performed using primers mF1 and AP1. The reaction solutioncontained 200 pM each of the primers, 0.1 mM each of dNTPs, 0.25 ml ofKlen Taq DNA Polymerase and the accompanied buffer to the polymerase tomake the total volume 25 ml. For amplification, 5 cycles of 98° C. for10 sec and 72° C. for 2 min; 5 cycles of 98° C. for 10 sec and 70° C.for 2 min; and 25 cycles of 98° C. for 10 sec and 68° C. for 2.5 minwere performed. Subsequently, using the first PCR reaction solution as atemplate, the second PCR was performed. The amplification of the5′-terminal region was performed with the same primer set as used forthe first PCR. The amplification of the 3′-terminal region was performedwith primers mF3 and AP1. The composition of the reaction solution wasthe same as in the reaction solution for the first PCR. Foramplification, 5 cycles of 98° C. for 10 sec and 72° C., for 2 min; 5cycles of 98° C. for 10 sec and 70° C. for 2 min; and 38 cycles of 98°C. for 10 sec and 68° C. for 2.5 min were performed.

[0756] Confirmation of the 5′-terminal and 3′-terminal amplifiedproducts were performed by 1.2% agarose gel electrophoresis and ethidiumbromide staining. PCR product bands were purified with QIA Quick GelExtraction Kit (Quiagen) and then sequenced. Determination of thenucleotide sequence was performed in the same manner as described inReference Example 3. Further, based on the resultant nucleotidesequence, the following two primers were synthesized. moF:5′-TTTAGACTTAGACGAAATGGA-3′ (SEQ ID NO: 31) moR:5′-GCTCCGTAGCCTCTTGAAGTC-3′ (SEQ ID NO: 32)

[0757] Using these primers and, as a template, the above-described cDNAsynthesized from mouse brain poly(A)⁺ RNA with SuperScript II RNase H—reverse transcriptase, PCR reaction was performed to amplify a fragmentcontaining the fill-length cDNA encoding a mouse-type physiologicallyactive peptide. The reaction was carried out with Klen Taq DNApolymerase (Clontech) under the conditions of 35 cycles of 98° C. for 10sec, 56° C. for 20 sec and 72° C. for 15 sec. The amplified product ofapproximately 600 bp was detected by 2% agarose gel electrophoresis andethidium bromide staining. The band was purified with QIA Quick GelExtraction Kit (Qiagen) and sub-cloned into cloning vector pCR2.1-TOPO(TOPO TA cloning kit; Invitrogen), which was then introduced into E.coli JM109 to thereby obtain transformant E. coli JM109/pmLP4. Thenucleotide sequence was determined in the same manner as in ReferenceExample 3. The nucleotide sequence determined (SEQ ID NO: 34), and theamino acid sequence deduced therefrom (SEQ ID NO: 33) are shown in FIG.7.

Reference Example 7

[0758] (1) Cloning of a cDNA Encoding G Protein-Coupled Receptor Proteinfrom the Peripheral Area of Rat Brainstem and Determination of theNucleotide Sequence thereof.

[0759] Using cDNA from the peripheral area of rat brainstem as atemplate, PCR reaction was performed with primer 1 (SEQ ID NO: 35) andprimer 2 (SEQ ID NO: 36). The composition of the reaction solution wasas follows: {fraction (1/10)} volume of the above cDNA as a template,{fraction (1/50)} volume of Advantage cDNA Polymerase Mix (Clontech),0.2 μM each of primer 1 (SEQ ID NO: 35) and primer 2 (SEQ ID NO: 36),200 μM dNTPs, and the accompanied buffer to the polymerase to make thereaction solution 50 μl. Thermal conditions of the PCR were as follows:(i) 94° C. for 2 min, then (ii) 3 cycles of 94° C. for 30 sec and 72° C.for 2 min, (iii) 3 cycles of 94° C. for 30 sec and 68° C. for 2 min,(iv) 30 cycles of 94° C. for 30 sec, 64° C. for 30 sec and 68° C. for 2min, and finally (v) extension at 68° C. for 8 min. The resultant PCRproduct was sub-cloned into plasmid vector pCR2.1 (Invitrogen) accordingto the protocol attached to TA cloning kit (Invitrogen). This vector wasintroduced into E. coli DH5α. From the resultant transformants, thoseclones carrying the cDNA were selected in ampicillin-containing LB agarmedium. As a result of analysis of the nucleotide sequences ofindividual clones, a cDNA sequence encoding a novel G protein-coupledreceptor protein was obtained (SEQ ID NO: 38). A novel G protein-coupledreceptor protein comprising the amino acid sequence (SEQ ID NO: 37)deduced from this cDNA sequence was designated rOT7T022L.

[0760] Plasmid pAK-rOT022L into which the cDNA (SEQ ID NO: 38) encodingthe G protein-coupled receptor protein rOT7T022L of the inventionderived from the peripheral area of rat brainstem had been sub-clonedwas introduced into Escherichia coli DH10B to thereby obtaintransformant Escherichia coli DH10B/rOT7T022L.

[0761] (2) Establishment of G Protein-Coupled Receptor ProteinrOT7T022L-Expressing CHO Cells

[0762] CHO dhfr⁻ cells (1×10⁶) were plated in a 10 cm tissue cultureplate and cultured for 24 hr. Using 20 μg of the rOT7T022L expressionvector obtained in (1) above and a gene transfer kit (Gen Transfer:Nippon Gene) utilizing the liposome method, a DNA/liposome complex wasformed. The medium was exchanged for fresh medium, to which theDNA/liposome complex was added and incubated overnight. After exchangeof the medium for fresh medium, cells were cultured further for one day.Then, the medium was changed for a transformant selection medium, andcells were cultured for two days in this medium. Further, cells weretreated with trypsin-EDTA and recovered from the tissue culture plate,and cells were re-cultured under a state of extremely low cell densityin order to increase the ratio of transformants. As a result, a cellclone CHO-rOT7T022L that expresses rOT7T022L highly and stably wasobtained.

[0763] (3) Synthesis ofMet-Pro-His-Ser-Phe-Ala-Asn-Leu-Pro-Leu-Arg-Phe-NH₂ (SEQ ID NO: 39)

[0764] Commercial p-methyl BHA resin (Applied Biosystems; current PerkinElmer) (0.5 mmole) was placed in a reactor in a peptide synthesizer(Applied Biosystems Model 430A), and swollen with DCM. Then, the firstamino acid Boc-Phe was activated by the HOBt/DCC method and introducedinto the p-methyl BHA resin. Subsequently, the resin was treated with50% TFA/DCM to remove the Boc group to thereby liberate the amino group,and then neutralized with DIEA. The subsequent amino acid Boc-Arg (los)was condensed with this amino group by the HOBt/DCC method. The presenceor absence of unreacted amino groups was examined by a ninhydrin testWhen the completion of the reaction was confirmed, Boc-Leu, Boc-Pro,Boc-Leu, Boc-Asn, Boc-Ala, Boc-Phe, Boc-Ser(Bzl), Boc-His(Bom), Boc-Proand Boc-Met were condensed in succession in the same manner as describedabove.

[0765] The resin into which the entire sequence of the amino acids hadbeen introduced was treated with 50% TFA/DCM to remove the Boc groups onthe resin, and then dried. As a result, 0.73 g ofMet-Pro-His(Bom)-Ser(Bzl)-Phe-Ala-Asn-Leu-Pro-Leu-Arg(Tos)-Phe-pMBHA-resinwas obtained.

[0766] In a hydrogen fluoride reactor made of Teflon, 0.25 g of thisresin was reacted with 5.1 g of p-cresol and 15 ml of hydrogen fluorideat 0° r for 60 minutes. The hydrogen was distilled off under reducedpressure. The residue was diluted with 100 ml of diethyl ether, stirred,filtered through a glass filter, and the fraction on the filter wasdried. This fraction was suspended in 50 ml of 50% aqueous solution ofacetic acid and stirred to extract the peptide. After the extraction,the resin was separated and the peptide was concentrated under reducedpressure to about 5 ml. This peptide was applied to a Sephadex G-25column (2×90 cm). Development was carried out with 50% aqueous aceticacid solution, and major fractions were collected and lyophilized.Subsequently, this crude peptide was dissolved in 1.5 ml of 5%thioglycollic acid/50% acetic acid and retained at 50° C. for 12 hr toreduce the Met-oxidant peptide. Then, the peptide was applied to areversed phase column packed with LiChroprep RP-18 (Merck) andrepeatedly purified by gradient elution using 0.1% TFA/H₂O and 0.1%TFA-containing 33% acetonitrileH₂O. Those fractions eluted at theacetonitrile concentration of around 27% were pooled and lyophilized toobtain 26 mg of white powder.

[0767] (M+H)⁺ value by mass spectrometry: 1428.7 (theoretical value:1428.8)

[0768] HPLC elution time: 18.0 min

[0769] Column conditions:

[0770] Column: Wakosil 5C18 (4.6×100 mm)

[0771] Eluent:

[0772] A (0.1% TFA-containing 5% acetonitrile/H₂O)

[0773] B (0.1% TFA-containing 55% acetonitrile/H₂O) Linear gradientelution from A to B (25 min.)

[0774] Flow rate: 1.0 ml/min

[0775] (4) Synthesis of Val-Pro-Asn-Leu-Pro-Gln-Arg-Phe-NH₂ (SEQ ID NO:40)

[0776] Boc-Phe, Boc-Arg(Tos), Boc-Gln, Boc-Pro, Boc-Leu, Boc-Asn,Boc-Pro and Boc-Val were condensed in succession in the same manner asdescribed in (3) in Reference Example 7, to thereby obtain 0.43 g ofBoc-Val-Pro-Asn-Leu-Pro-Gln-Arg(Tos)-Phe-pMBHA-resin. This resin (0.22g) was treated with hydrogen fluoride and purified by columnchromatography in the same manner as described above to thereby obtain46 mg of desired white powder.

[0777] (M+H)⁺ value by mass spectrometry: 969.5 (theoretical value:969.6)

[0778] HPLC elution time: 11.8 min

[0779] Column conditions: Column: Wakosil 5C18(4.6×1001 mm)

[0780] Eluent:

[0781] A (0.1% TFA-containing 5% acetonitrile/H₂O)

[0782] B (0.1% TFA-containing 55% acetonitrile/H₂O)

[0783] Linear gradient elution from A to B (25 min.)

[0784] Flow rate: 1.0 ml/min

[0785] (5) Synthesis of Ser-Ala-Gly-Ala-Thr-Ala-Asn-Leu-Pro-Arg-Ser-NH₂(SEQ ID NO: 41)

[0786] Boc-Ser(Bzl), Boc-Arg(Tos), Boc-Leu, Boc-Pro, Boc-Leu, Boc-Asn,Boc-Ala, Boc-Thr(Bzl), Boc-Ala, Boc-Gly, Boc-Ala and Boc-Ser(Bzl) werecondensed in succession in the same manner as described in (3) inReference Example 7, to thereby obtain 0.62 g ofBoc-Ser(Bzl)-Ala-Gly-Ala-Thr(Bzl)-Ala-Asn-Leu-Pro-Leu-Arg(Tos)-Ser(Bzl)-pMBHA-resin.This resin (0.23 g) was treated with hydrogen fluoride and purified bycolumn chromatography in the same manner as described above to therebyobtain 71 mg of desired white powder.

[0787] (M+H)⁺ value by mass spectrometry: 1156.4 (theoretical value:1156.6)

[0788] HPLC elution time: 11.8 mm

[0789] Column conditions:

[0790] Column: Wakosil 5C18 (4.6×100 mm)

[0791] Eluent:

[0792] A (0.1% TFA-containing 5% acetonitrile/H₂O)

[0793] B (0.1% TFA-containing 55% acetonitrile/H₂O)

[0794] Linear gradient elution from A to B (25 min.)

[0795] Flow rate: 1.0 ml/min

[0796] (6) Reaction Experiment between rOT7T022L (SEQ ID NO: 37) andPeptide MPHSFANLPLRF amide (SEQ ID NO: 39) and Between rOT7T022L andPeptide VPNLPQRF amide (SEQ ID NO: 40) Using a Cytosensor

[0797] The rOT7T022L receptor-expressing CHO cells obtained in (2) inReference Example 7 were sown in cytosensor capsules at 2.7×10⁵cells/capsule. After overnight culture, the capsules were mounted in thework station of a cytosensor. The assay medium (0.1% bovine serumalbumin-containing low buffered RPMI1640 medium) positioned in the flowroute of the cytosensor was supplied to the cells under pump ON (80sec)/pump OFF (40 sec) cycles. In each cycle, the change ratio ofextracellular pH for 30 sec starting from 8 sec after the stop of thepump was calculated as acidification rate. The time course ofacidification rate was monitored. When this rate began to show stablevalues, the flow route was shifted to thereby expose individual peptideto the cells for 7 min and 2 sec. The values of acidification rate inindividual wells were normalized taking the values for the 3 cyclesimmediately before the peptide exposure as 100%. Comparison of thereactions of the cells revealed that rOT7T022L receptor-expressing CHOcells were reactive with peptide MPHSFANLPLRF amide (SEQ ID NO: 39) andpeptide VPNLPQRF amide (SEQ ID NO: 40) in a strongly dose dependentmanner (FIG. 8).

Reference Example 8 Preparation of Transformant Carrying a CandidateSplicing Variant cDNA Encoding a Human Novel Physiologically ActivePeptide

[0798] The reaction product obtained in the PCR performed in ReferenceExample 3 above was separated with 1.2% agarose gel to thereby confirmthe amplification of a DNA fragment of the expected size. Then, the DNAwas recovered with Qiagen PCR Purification Kit (Quiagen). According tothe protocol attached to a TA cloning kit (Invitrogen), the recoveredDNA was subcloned into plasmid vector pCR™2.1. This plasmid vector wasintroduced into E. coli JM109 competent cells (Takara Shuzo) to preparetransformants. From the resultant transformants, those clones carryingthe inserted cDNA fragment were selected in LB agar medium containingampicillin, IPTG and X-gal. Only those clones presenting a white colorwere isolated with a sterilized tooth pick. Each of these clones wascultured overnight in ampicillin-containing LB medium and then plasmidDNA was prepared therefrom using an automated plasmid extractionapparatus (Kurabo). A part of the thus prepared DNA was digested withEcoRI, followed by confirmation of the size of the inserted cDNAfragment. A part of the remaining DNA was treated with RNase, extractedwith phenol:chloroform, and then precipitated with ethanol forconcentration. Reactions for determining the nucleotide sequence wereperformed with Dye Deoxy Terminator Cycle Sequencing Kit (ABI), followedby reading with a fluorescence-based automated sequencer. As a result,transformant Escherichia coli JM109/phRF2 was obtained.

Reference Example 9 Preparation of Transformant Carrying a cDNA EncodingBovine Novel Physiologically Active Peptide

[0799] Using 1 ml of the bovine hypothalamus cDNA prepared in ReferenceExample 4, PCR amplification was performed with the following twoprimers bFF and bFR. bFF: 5′-TTCTAGATTTTGGACAAAATGGAAATT-3′ (SEQ ID NO:52) bFR: 5′-CGTCTTTAGGGACAGGCTCCAGATTTC-3′ (SEQ ID NO: 53)

[0800] The composition of the reaction solution was as follows: 20 pMeach of synthetic primers (bFF and bFR), 0.25 mM dNTPs, 0.5 ml of Ex TaqDNA polymerase and the accompanied buffer to the polymerase to make thereaction solution 50 ml. The amplification was performed in a thermalcycler (Perkin Elmer) under the following conditions: 40 cycles of 98°C. for 10 sec, 65° C. for 20 sec and 72° C. for 20 sec. The amplifiedproduct was confirmed by 1.2% agarose gel electrophoresis and ethidiumstaining. The reaction product obtained in the PCR performed inReference Example 3 above was separated with 1.2% agarose gel to therebyconfirm the amplification of a DNA fragment of the expected size. Then,the DNA was recovered with Qiagen PCR Purification Kit (Quiagen).According to the protocol attached to a TA cloning kit (Invitrogen), therecovered DNA was subcloned into plasmid vector pCR™2.1. This plasmidvector was introduced into E. coli JM109 competent cells (Takara Shuzo)to prepare transformants. From the resultant transformants, those clonescarrying the inserted cDNA fragment were selected in LB agar mediumcontaining ampicillin, IPTG and X-gal. Only those clones presenting awhite color were isolated with a sterilized tooth pick. Each of theseclones was cultured overnight in ampicillin-containing LB medium andthen plasmid DNA was prepared therefrom using an automated plasmidextraction apparatus (Kurabo). A part of the thus prepared DNA wasdigested with EcoRI followed by confirmation of the size of the insertedcDNA fragment. Further, the prepared DNA was treated with RNase,extracted with phenol:chloroform, and then precipitated with ethanol forconcentration. Reactions for determining the nucleotide sequence wereperformed with Dye Deoxy Terminator Cycle Sequencing Kit (ABI), followedby reading with a fluorescence-based automated sequencer. As a result,transformant Escherichia coli JM109/pbRF2 was obtained.

Reference Example 10 cAMP Production Inhibitory Effects of PeptideMPHSFANLPLRF amide (SEQ ID NO: 39) and Peptide VPNLPQRF Amide (SEQ IDNO: 40) Upon rOT7T022L (SEQ ID NO: 37)-Expressing CHO Cells

[0801] The experiment using a cytosensor conducted in (6) in ReferenceExample 7 confirmed that peptides MPHSFANLPLRF amide (SEQ ID NO: 39) andVPNLPQRF amide (SEQ ID NO: 40) synthesized in (3) and (4) in ReferenceExample 7, respectively, react with rOT7T022L receptor specifically.Subsequently, the cAMP production inhibitory effects of these peptidesupon rOT7T022L-expressing CHO cells were measured.

[0802] The rOT7T022L-expressing CHO cells obtained in (2) in ReferenceExample 7 were plated in 24-well plated at 1.0×10⁵ cells/well andcultured at 37° C. for 2 days. Cells were washed with Hanks' buffersupplemented with 0.05% BSA and 0.2 mM IBMX, and then left in the samebuffer at 37° C. for 30 min. Thirty minutes later, an assay buffer (theabove-mentioned buffer to which 106 M forskolin is added) and theabove-mentioned peptides at varied concentrations were added to thecells, followed by incubation at 37° C. for 30 min.

[0803] Thirty minutes later, intracellular cAMP concentrations inindividual wells were measured according to the protocol attached to thecAMP EIA Kit (Amersham). As a result, as shown in FIG. 9, peptidesMPHSFANLPLRF amide (SEQ ID NO: 39) and VPNLPQRF amide (SEQ ID NO: 40)revealed cAMP production inhibitory effects upon rOT7T022Lreceptor-expressing CHO cells. The IC₅₀ values were 0.5 nM and 0.7 nM,respectively; thus, these peptides revealed strong effects at very lowconcentrations.

Reference Example 11

[0804] (1) Cloning of a cDNA Encoding G Protein-Coupled Receptor Proteinfrom Human Hypothalamus and Determination of the Nucleotide SequenceThereof

[0805] PCR reaction was performed using human hypothalamus cDNA(Clontech) as a template and the following two primers: primer 1:5′-GTCGACATGG AGGGGGAGCC CTCCCAGCCT C-3′ (SEQ ID NO: 57) and primer 2:5′-ACTAGTTCAG ATATCCCAGG CTGGAATGG-3′ (SEQ ID NO: 58). The compositionof the reaction solution was as follows: {fraction (1/10)} volume of theabove-mentioned cDNA as a template, {fraction (1/50)} volume ofAdvantage-HF Polymerase Mix (Clontech), 0.2 μM each of primer 1 (SEQ IDNO: 57) and primer 2 (SEQ ID NO: 58), 200 μM dNTPs, 4% dimethylsulfoxide, and the accompanied buffer to the polymerase to make thereaction solution 25 μl. Thermal conditions of the PCR were as follows:(i) 94° C. for 2 min, then (ii) 3 cycles of 94° C. for 20 sec and 72° C.for 1.5 min, (iii) 3 cycles of 94° C. for 20 sec and 67° C. for 1.5 min,(iv) 38 cycles of 94° C. for 20 sec., 62° C. for 20 sec and 72° C./68°C. for 1.5 min, and finally (v) extension at 68° C. for 7 min. Theresultant PCR product was sub-cloned into plasmid vector pCR2.1(Invitrogen) according to the protocol attached to TA cloning kit(Invitrogen). This vector was introduced into E. coli DH5α. From theresultant transformants, those clones carrying the cDNA were selected inampicillin-containing LB agar medium. As a result of analysis of thenucleotide sequences of individual clones, cDNA sequences encoding anovel G protein-coupled receptor protein were obtained (SEQ ID NOS: 55and 56). Although these two sequences are different from each other inone nucleotide at position 597, amino acid sequences deduced therefromare the same (SEQ ID NO: 57). A novel G protein-coupled receptor proteincomprising this amino acid sequence was designated hOT7T022. The twotransformants were designated Escherichia coli DH5 α/pCR2.1-hOT022T(carrying the cDNA represented by SEQ ID NO: 55) and Escherichia coliDH5α/pCR2.1-hOT022G (carrying the cDNA represented by SEQ ID NO: 56),respectively.

Reference Example 12 Preparation of Anti-Rat RFRP-1 Monoclonal Antibody

[0806] A monoclonal antibody was prepared against a peptideC-VPHSAANLPLRF-NH₂ as an antigen; this peptide represents the C-terminal12 amino acids of rat-type RFRP-1 (the C-terminal carboxyl group isamidated: an amino acid sequence which is from position 83 (Val) toposition 94 (Phe) of the amino acid sequence of SEQ ID NO: 50) to whichone Cys residue is added at the N-terminus. The antigen peptide (0.6 mg)was conjugated with bovine serum albumin (BSA) using maleimide. Theresultant conjugate (100 μg) was subcutaneously injected into mice threetimes to immunize them. Then, 50 μg of the conjugate was injected intothe tail vein as final immunization. Four days after the finalimmunization, splenocytes were recovered from each mouse and fused tomouse myeloma cells (P3-X63Ag8-U1; Matsumoto et al, BBRC (1999) vol.257, 264-268) using polyethylene glycol. After this cell fusion, ahybridoma cell 1F3 was selected and cultured in a large quantity usingINTREGRA CL-1000 to thereby obtain a culture supernatant of IF3. Fromthis culture supernatant, anti-rat RFRP-1 monoclonal antibody wasobtained using HiTrap rProtein A column (Pharmacia). Isotyping of thismonoclonal antibody with Mouse mAb Isotyping Kit (Amersham) revealedthat the subtype of this monoclonal antibody was IgG1 κ chain.

Reference Example 13 Construction of Competitive EIA

[0807] First, the peptide used as the antigen in Reference Example 12was conjugated with horse radish peroxidase (HRP) using maleimide tothereby prepare a conjugate HRP-rat RFRP-1. Then, using this conjugateHRP-rat RFRP-1 and the anti-rat RFRP-1 monoclonal antibody obtained inReference Example 12, a competitive EIA was constructed.

[0808] Briefly, 50 μl of the anti-rat RFRP-1 monoclonal antibody dilutedwith a buffer (phosphate buffered saline (PBS) containing 2 mM EDTA,0.4% BSA, 0.1 M NaCl and 0.1% micro-O-protect) was added to each well of96-well plates coated with anti-mouse IgGAM (Cappel) at 1.5 μg/well andblocked with Block ACE (Dainippon Pharmaceutical). A 50 ml sampledissolved in the same buffer was also added to each well. After a 16-hrincubation at 4° C., 50 μl of HRP-rat RFRP-1 diluted with the buffer wasadded to each well. After 2-hr incubation at room temperature, theplates were washed with 0.1% Tween 20 (Sigma)-containing PBS. Then, theactivity of IRP bound to each well was detected by a color reactionusing TMB microwell peroxidase system (Kirkegaard & Perry Labs),followed by measurement of the absorbance at 450 nm. Changes inabsorbance when RFRP-1-related peptides were added are shown in FIG. 11.

Example A1 Effects of Ligand Peptide upon Plasma Pituitary HormoneLevels

[0809] Effects of the administration of the peptide represented by SEQID NO: 39 into the third ventricle upon pituitary hormone levels wereexamined. Briefly, adult male Wistar rats (body weight: 290-350 g at thetime of surgery) were anesthetized by intraperitoneal administration of50 mg/kg pentobarbital and fixed in a stereotaxic apparatus for ratbrain. The incisor bar was set 3.3 mm below the interaural line. Theskull surface was exposed and a hole was drilled in the skull with adental drill in order to implant a guide cannula Further, an anchorscrew was embedded at one place around the hole. A stainless guidecannula AG-12 (inside dia. 0.4 mm, outside dia. 0.5 mm; Eicom) wasinserted so that its tip is located above the third ventricle. Thestereotaxic coordinates were set as follows according to the atlas ofPaxinos and Watson (1998): AP: +7.2 mm (from the interaural line), L:0.0 mm, H: +2.0 mm (from the interaural line). The guide cannula wasfixed onto the skull with instant glue, dental cement and the anchorscrew. A stainless dummy cannula AD-12 (outside dia. 0.35 mm; Eicom) wasinserted into the guide cannula and fixed with a cap nut (Eicom). Afterthe surgery, the rats were bred separately in individual cages for morethan one week to allow recovery.

[0810] The day before the experiment, the rats that had undergone theabove surgery were anesthetized by intraperitoneal administration of 50mg/kg pentobarbital and fixed on anatomical pads in face-up position.Then, a catheter (SP35; Natsume Seisakusho) was inserted into the rightjugular vein. The next day, 400 μl of blood was taken from the jugularvein. In order to prevent blood coagulation, 20 μl of physiologicalsaline containing 200 units/ml heparin was put in advance in theinjection syringe. The cap nut and the dummy cannula installed on therat skull were removed. Instead of them, a stainless microinjectioncannula AMI13 (inside dia. 0.17 mm, outside dia. 0.35 mm; Eicom)connected with a Teflon tube (length 50 cm, inside dia. 0.1 mm, outsidedia. 0.4 mm; Eicom) was inserted into the guide cannula. The length ofthe microinjection cannula was adjusted in advance so that 1 mm of itstip is exposed from the guide cannula. One end of the Teflon tube wasconnected with a microsyringe pump. Then, a total 10 μl of PBS alone orPBS containing the peptide represented by SEQ ID NO: 39 was injectedinto the third ventricle at a flow rate of 5 μl/min. One minute afterthe completion of the injection, the microinjection cannula was removed,and a dummy cannula was fixed again with a cap nut. A 400 μl bloodsample was taken from the cannula inserted into the jugular veinimmediately before the start of the intraventricular administration andat 10, 20, 30, 40 and 60 min after the start of the intraventricularadministration. The collected blood was centrifuged at 5,000 rpm for 10min in a high-speed refrigerated microcentrifuge (MR-150; Tomy Seiko) torecover the supernatant (plasma). The prolactin levels in the plasmawere measured by radioimmunoassay.

[0811] The results are expressed as mean±S.E.M. Whether there is asignificant difference between the group to which PBS containing thepeptide represented by SEQ ID NO: 39 was administered and the controlgroup to which PBS alone was administered was tested by Student'st-test. Differences were assessed with two-sided tests, and significancelevel 5% or below was judged statistically significant. As shown in FIG.10, plasma prolactin levels showed an increasing tendency from 10 minafter the administration of 10 mnol peptide represented by SEQ ID NO: 39into the third ventricle; the levels increased significantly at 20, 30and 40 min after the administration. Even 60 min after theadministration, significant difference was observed between theadministration group and the control group. The GH, LH, ACTH and TSHlevels in the plasma did not show significant changes.

Example A2 Purification of Endogenous RFRP-1 from Bovine Hypothalamus

[0812] RFRP-1-like immune activity was found in a crude peptide fractionfrom bovine hypothalamus in the competitive EIA constructed in ReferenceExample 13. Using this RFRP-1-like immune activity as an indicator, anendogenous RFRP-1 was purified from bovine hypothalamus.

[0813] First, 2.0 kg of frozen bovine hypothalamus was boiled inultra-pure water (milli-Q water), to which acetic acid was added to givea concentration of 1M. Then, the hypothalamus was homogenized with aPolytron. After overnight stirring, supernatant was obtained bycentrifugation. Trifluoroacetic acid (TFA) was added to the supernatantto give a concentration of 0.05%, and the supernatant was applied to aC18 column (Prep C18 125 A; Waters). The peptide bound to the column waseluted in a step-wise manner with 0.05% TFA-containing 10%, 30% and 50%acetonitrile solution. 30% acetonitrile fraction was diluted with twovolumes of 20 mM ammonium acetate (pH 4.7) and applied to an ionexchange column HiPrep CM-Sepharose FF (Pharmacia). The peptide bound tothe ion exchange column was eluted in a step-wise manner with 0.1, 0.2,0.5 and 1.0 M NaCl in 10% acetonitrile-containing 20 mM ammonium acetate(pH 4.7). Three volumes of cold acetone was added to 0.1 M NaCl fractionwhich contained the highest RFRP-1-like immune activity, followed bycentrifugation to remove the precipitate. The resultant supernatant wasconcentrated in an evaporator. TFA was added to the concentratedsupernatant to give a concentration of 0.1%, and then the supernatantwas applied to a reversed-phase HPLC column RESOURCE RPC (Pharmacia) forfurther separation. Elution from the RESOURCE RPC column was performedwith the density gradient of 10-30% acetonitrile. The major RFRP-1-likeactivity was eluted with approximately 22% acetonitile. This activefraction was separated in a cation exchange column TSK gel CM-SW (Tosoh)using the density gradient of 0.2-0.6 M NaCl in 10%acetonitrile-containing 20 mM ammonium acetate (pH 4.7). The majorRFRP-1-like activity was eluted with approximately 0.3 M NaCl. TFA wasadded to the RFRP-1-like activity-containing CM-2SW column fraction togive a concentration of 0.1%. Then, this fraction was fractionatedfurther in a reversed-phase column Diphenyl 219TP52 (Vydac). As a resultof elution using the density gradient of 21-25% acetonitrile,RFRP-1-like immune activity was eluted with 23% acetonitrile. ThisRFRP-1-like immune activity-containing fraction was purified finally ina reversed-phase column u RPC C2C18 SC2.1/10 using the density gradientof 22-23% acetonitile to thereby obtain a single peak which isconsistent with the RFRP-1-like immune activity (FIG. 12).

Example A3 Analysis of the N-Terminal Amino Acid Sequence of the FinallyPurified Product and Determination of its Molecular Weight by MassSpectrometry

[0814] When N-terminal amino acids of the finally purified productobtained in Example A3 were analyzed with a protein sequencer (Model491cLC; Applied Biosystems), an amino acid sequenceS-L-T-F-E-E-V-K-D-X-A-P-K-1-K-M-N-K-P-V- (where X represents anunidentified amino acid residue) was obtained.

[0815] When the molecular weight of the finally purified product wasdetermined with ESI-MS (Mermoquest), a value 3997.0 was obtained.

[0816] From these results, it was found that the finally purifiedproduct from bovine hypothalamus is a peptide consisting of the 35 aminoacids from position 58 (Ser) to position 92 (Phe) of the amino acidsequence represented by SEQ ID NO: 14.

Example A4 Preparation of a Structural Gene for a Peptide which has anAmino Acid Sequence Which is from Position 56 (Ser) to Position 92 (Phe)of the Amino Acid Sequence Represented by SEQ ID NO: 1 and WhoseC-Terminal Carboxyl Group is Amidated (hereinafter, Sometimes Referredto as “hRFRIP-1(37)”)

[0817] A structural gene for hRPRP-1(37) was prepared using the four DNAfragment shown in SEQ ID NOS: 59-62 (#1: SEQ ID NO: 15 and #4: SEQ IDNO: 18; Kiko-Tech) and (#2: SEQ ID NO: 16 and #3: SEQ ID NO: 17;Amersham Pharmacia Biotech).

[0818] a) Phosphorylation of DNA Oligomers

[0819] Two oligomers #1 and #4 which are to be the 5′-terminal regionswere excluded from this treatment. One microgram each of the remainingtwo oligomers was reacted in 100 μl of phosphorylation reaction solution[50 mM Tris-HCl (pH 7.6), 10 mM MgCl₂, 1 mM spermidine, 10 mMdithiothreitol, 0.1 mg/ml bovine serum albumin, 1 mM ATP, 10 units of T4polynucleotide kinase (Nippon Gene)] at 37° C. for 1 hr to phosphorylatethe 5′ end. After phenol treatment, the aqueous layer was recovered, andtwo volumes of ethanol was added thereto. The mixture was cooled to −70°C. and then centrifuged to precipitate DNA.

[0820] b) Ligation of DNA Fragments

[0821] The phosphorylated DNA fragments obtained in a) above and #1 and#4 (1 μg each) were added together to 10 mM Tris-HCl, 2 mM EDTA to makea 120 μl mixture. This mixture was retained at 80° C. for 10 min andthen gradually cooled to room temperature for annealing. Ligation wasperformed with TaKaRa DNA Ligation Kit ver. 2 (Takara Shuzo). Briefly,30 μl of liquid II contained in the kit was added to 30 μl of the aboveannealing mixture and mixed thoroughly. Then, 60 μl of liquid Icontained in the kit was added thereto and reacted at 37° C. for 1 hr toperform ligation. After phenol treatment, the aqueous layer wasrecovered, and two volumes of ethanol was added thereto. The mixture wascooled to −70° C. and then centrifuged to precipitate DNA.

[0822] c) Phosphorylation of the 5′ End

[0823] The precipitate was dissolved in 10 μl of TE buffer (10 mMTris-HCl (pH 8.0), 1 mM EDTA) and reacted in 100 μl of phosphorylationreaction solution [50 mM Tris-HCl (pH 7.6), 10 mM MgCl₂, 1 mMspermidine, 10 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 1 mMATP, 10 units of T4 polynucleotide kinase (Nippon Gene)] at 37° C. for 1hr to phosphorylate the 5′ end. After phenol treatment, the aqueouslayer was recovered, and two volumes of ethanol was added thereto. Themixture was cooled to −70° C. and then centrifuged to precipitate DNA,which was dissolved in 20 μl of TE buffer.

Example A5 Preparation of hRFRP-1(37) Expression Plasmid

[0824] As an expression vector, pTFC (described in Japanese UnexaminedPatent Publication No. 2000-270871) was digested with NdeI and AvaI(Takara Shuzo) at 37° C. for 4 hr. The digest was electrophoresed on 1%agarose gel, and a 4.4 kb DNA fragment was extracted with QIAquick GelExtraction Kit (Qiagen) and dissolved in 25 μl of TE buffer. ThisNdeI-AvaI fragment from pTFC was ligated to the structural gene forhRFRP-1(37) prepared above using TaKaRa DNA Ligation Kit ver. 2 (TakaraShuzo). With 10 μl of the resultant ligation reaction solution, E. coliJM109 competent cells were transformed and plated on 10 μg/mltetracycline-containing LB agar medium. After overnight culture at 37°C., tetracycline resistant colonies grown on the medium were selected.This transformant was cultured overnight in LB medium, followed bypreparation of plasmid pTFCRFRP-1 using QIAprep8 Miniprep Kit (Qiagen).The nucleotide sequence of the structural gene moiety for hRFRP-1(37) inthis plasmid was confirmed with Model 377 DNA Sequencer (AppliedBiosystems). E. coli MM294 (DE3) was transformed with plasmid pTFCRFRP-1to thereby obtain RFRp-1-CS23 fusion protein-expressing Escherichia coliMM294 (DE3)/pTFCRFRP-1 (FIG. 13).

Example A6

[0825] The transformant obtained in Example A5 was cultured undershading in 1 L of 5.0 mg/L tetracycline-containing LB medium (1%peptone, 0.5% yeast extract, 0.5% sodium chloride) in a 2 L-flask at 37°C. for 8 hr. The resultant culture broth was transferred into a 50L-fermenter containing 19 L of primary fermentation medium (1.68% sodiummonohydrogenphosphate, 0.3% potassium dihydrogenphosphate, 0.1% ammoniumchloride, 0.05% sodium chloride, 0.05% magnesium sulfate, 0.02%defoaming agent, 0.00025% ferrous sulfate, 0.00025% thiaminehydrochloride, 1.5% glucose, 1.5% casamino acid) and cultured at 30° C.under aeration and agitation. When the turbidity of the culture brothreached about 500 Klett Units, isopropyl-β-D-thiogalactopyranoside wasadded to give a final concentration of 12 mg/L. Cells were cultured foranother 4 hr. After completion of the cultivation, the culture broth wascentrifuged to thereby obtain about 500 g of wet cells, which werefrozen and stored at −80° C.

Example A7 Acquisition of hRFRP-1(37)

[0826] To the 500 g of the cells obtained in Example A6, 1000 ml of asolution containing 6 M guanidine hydrochloride and 0.2 M Tris-HCl (pH8.0) was added and agitated for about 4 hr. The resultant mixture wascentrifuged (10000 rpm, 60 min), and the supernatant was diluted with 29L of a solution containing 1 mM dithiothreitol and 50 mM Tris-HCl (pH8.0). The diluted solution was left stationary overnight at 10° C. Then,the pH of the solution was adjusted at 6.0 with concentrated HCl. Theresultant solution was fed to an AF-Heparin Toyopearl 650M column (11.3cm ID×13 cm L; Tosoh) pre-equilibrated with 50 mM phosphate buffer (pH6.0). After adsorption and washing, the adsorbate was eluted with 50 mMphosphate buffer, 2M NaCl, pH 6.0. As a result, 1000 ml ofhRFRP-1(37)-CS23 fusion protein (i.e. a fusion protein of thepolypeptide of the invention and CS23) fraction was obtained.

[0827] This eluate was concentrated in Pellicon Minicassette (Millipore)while adding 0.1 M acetic acid. As a result, a solution ofhRFRP-1(37)-CS23 fusion protein in 0.1 M acetic acid was obtained. Ureawas added to this solution to give a final concentration of 6M, and then445 mg of 1-cyano-4-dimethylamino pyridinium salt (DMAP-CN) was addedthereto and reacted at room temperature for 15 min. After completion ofthe reaction, the reaction solution was fed to a Sephadex G-25 column(46 mm ID×600 mm L; Pharmacia) pre-equilibrated with 10% acetic acid.Then, 10% acetic acid was fed to the column at a flow rate of 6 nil/minto thereby obtain a fraction of S-cyanylated hRFRP-1(37)-CS23 fusionprotein. This eluate was concentrated and de-salted in PelliconMinicassette (Millipore) to thereby obtain a de-salted solution ofhRFRP-1(37)CS23 fusion protein. Urea was added to this de-saltedsolution to give a final concentration of 6 M. Further, 25% aqueousammonia was added thereto to give a concentration of 3 M. Then, theresultant solution was reacted at 15° C. for 15 min. After completion ofthe reaction, the pH of the reaction solution was adjusted at 6.0 withacetic acid to thereby obtain hRFRP-1(37). The reaction solution was fedto a SP-5PW column (5.5 cm ID×30 cm L; Tosoh) pre-equilibrated with 50mM MES buffer containing 3 M urea (pH 4.5). After adsorption andwashing, elution was performed with step-wise gradient of 0-50% solutionB (solution B=50 mM MES buffer+1 M NaCl+3 M urea) to thereby obtainhRFRP-1(37) (elution time: 60 min). This hRFRP-1(37) fraction wasfurther fed to an ODS-120T column (21.5 mm ID×300 mm L; Tosoh)pre-equilibrated with 0.1% trifluoroacetic acid (TFA). After adsorptionand washing, elution was performed with stepwise gradient of 3060%solution B (solution B=80% acetonitrile/0.1% TFA) to thereby poolhRFRP-1(37) fractions (elution time: 45 min). Then, these fractions werelyophilized to obtain lyophilized powder of hRFRP-1(37).

Example A8 Comparison of the Agonist Activities of Various RF AmidePeptides upon Human O07T022 Receptor-Expressing CHO Cells

[0828] Human OT7T022 receptor-expressing CHO cells prepared based on theprocedures described in WO 00/29441 were plated in 24-well plates at3×10⁵ cells/well and cultured overnight. The cells were washed withHanks' buffer (HBSS) supplemented with 0.05% BSA and 0.2 mM IBMX andthen preincubated in the same buffer at 37° C. for 30 min. Then, thebuffer was exchanged for HBSS supplemented with 0.05% BSA and 0.2 mMIBMX, this buffer supplemented with 1 μM forskolin alone, or this buffersupplemented with 1 μM forskolin and a peptide at a variedconcentration, and the cells were incubated at 37° C. for 30 min. Afterthe incubation, intracellular cAMP was extracted and determined for eachwell according to the protocol attached to the cAMP EIA Kit (Amersham).For each concentration of the peptides used, the ratio of inhibition ofintracellular cAMP increase by forskolin treatment was calculated tothereby obtain the dose-reaction curves as shown in FIG. 14. The ED₅₀values of the peptides tested were as follows: hRFRP-1-12 (peptidehaving the amino acid sequence which is from position 81 (Met) toposition 92 (Phe) of SEQ ID NO: 1, (◯)) (4.5 nM); hRFRP-1-37 (peptidehaving the amino acid sequence which is from position 56 (Ser) toposition 92 (Phe) of SEQ ID NO: 1, (▪)) (21 nM); rRFRP-1-37 (peptidehaving the amino acid sequence which is from position 58 (Ser) toposition 94 (Phe) of SEQ ID NO: 50, (⋄)) (30 nM); hRFRP-2-12 (peptidehaving the amino acid sequence which is from position 101 (Phe) toposition 112 (Ser) of SEQ ID NO: 1, (▴)); hRFRP-3-8 (peptide having theamino acid sequence which is from position 124 (Val) to position 131(Phe) of SEQ ID NO: 1, (□)) (9.9 nM); PQRF amide (peptide represented byPro-Gln-Arg-Phe-NH₂ (♦)) (1000 nM or more); LPLRF amide (peptiderepresented by Leu-Pro-Leu-Arg-Phe-NH₂ ()) (36 mM); and NPFF (peptiderepresented by Asn-Pro-Phe-Phe (Δ)) (140 nM).

Example A9 Examination of the Effect of Pertussis Toxin upon theActivation of Human OT7T022 Receptor by an RFRP Peptide

[0829] The human O07T022 receptor-expressing CHO cells obtained inExample A8 were plated in 24-well plates at 1×10⁵ cells/well andcultured overnight. Then, the medium was changed for 100 ng/nm pertussistoxin (Sigma)-containing medium or a control medium, and the cells werecultured overnight. The cells were washed with Hanks' buffer (HBSS)supplemented with 0.05% BSA and 0.2 mM IBMX and then pre-incubated inthe same buffer at 37° C. for 30 min. Subsequently, the buffer wasexchanged for HBSS supplemented with 0.05% BSA and 0.2 mM IBMX, HBSSsupplemented with 1 μM forskolin alone, or HBSS supplemented with 1 μMforskolin and a peptide at various concentrations, and the cells wereincubated at 37° C. for 30 min. After the incubation, the buffer wasexchanged for HBSS supplemented with 0.05% BSA and 0.2 mM IBMX (blackcolumn), this buffer supplemented with 1 μM forskolin alone (whitecolumn), or this buffer supplemented with 1 μM forskolin and 0.1 μMRFRP-1-12 (peptide having the amino acid sequence which is from position81 (Met) to position 92 (Phe) of SEQ ID NO: 1) (column with slantlines), and the cells were incubated at 37° C. for 30 min. After theincubation, intracellular cAMP was extracted and determined for eachwell according to the protocol attached to the cAMP EIA Kit (Amersham).As a result, as shown in FIG. 15, cAMP production inhibitory activitydisappeared in those cells treated with pertussis toxin. Thus, it wasshown that the cAMP production inhibitory reaction mediated by OT7T022receptor is co-operating with pertussis toxin sensitive G protein αsubunit Gi (inhibitory) or Go.

Example B1 Preparation of Anti-Rat RFRP-3 Monoclonal Antibody

[0830] A monoclonal antibody was prepared against a peptideC-FPSLPQRF-NH₂ as an antigen; this peptide represents the C-terminal 8amino acids of rat-type RFRP-3 (the C-terminus is amidated) to which oneCys residue is added at the N-terminus. The antigen peptide (0.6 mg) wasconjugated with bovine serum albumin (BSA) using maleimide. Theresultant conjugate (100 μg) was subcutaneously injected into mice threetimes to immunize them. Then, 50 μg of the conjugate was injected intothe tail vein as final immunization. Four days after the finalimmunization, splenocytes were recovered from each mouse and fused tomouse myeloma cells (P3-X63Ag8-U1) using polyethylene glycol. After thiscell fusion, a hybridoma cell 7F6 was selected and cultured in a largequantity using INTREGRA CL-1000 to thereby obtain a culture supernatantof 7F6. From this culture supernatant, anti-rat RFRP-3 monoclonalantibody was obtained using HiTrap rProtein A column (Pharmacia).Isotyping of this monoclonal antibody with Mouse mAb Isotyping Kit(Amersham) revealed that the subtype of this monoclonal antibody wasIgG2b κ chain.

Example B2 Construction of Competitive EIA

[0831] First, the peptide used as the antigen was conjugated with horseradish peroxidase (HRP) using maleimide to thereby prepare a conjugateHRP-rat RFRP-3. Then, using this conjugate HRP-rat RFRP-3 and theanti-rat RFRP-3 monoclonal antibody obtained in Example B1, acompetitive EIA was constructed.

[0832] Briefly, 50 μl of the anti-rat RFRP-3 monoclonal antibody dilutedwith a buffer (phosphate buffered saline (PBS) containing 2 mM EDTA,0.4% BSA, 0.1 M NaCl and 0.1% micro-O-protect) was added to each well of96-well plates coated with anti-mouse IgGAM (Cappel) at 1.5 μg/well andblocked with Block ACE (Dainippon Pharmaceutical). A 50 μl sampledissolved in the same buffer was also added to each well. After a 16-hrincubation at 4° C., 50 μl of HRP-rat RFRP-3 diluted with the buffer wasadded to each well. After 2-hr incubation at room temperature, theplates were washed with 0.1% Tween 20 (Sigma)-containing PBS. Then, theactivity of HRP bound to each well was detected by a color reactionusing TMB microwell peroxidase system, followed by measurement of theabsorbance at 450 nm. Changes in absorbance when RFRP-3-related peptideswere added are shown in FIG. 16.

Example B3 Purification of Endogenous RFRP-3 from Bovine Hypothalamus

[0833] RFRP-3-like immune activity was found in a crude peptide fractionfrom bovine hypothalamus in the competitive EIA constructed in ExampleB2. Using this RFRP-3-like immune activity as an indicator, anendogenous RFRP-3 was purified from bovine hypothalamus.

[0834] First, 2.0 kg of frozen bovine hypothalamus was boiled inultra-pure water (milli-water), to which acetic acid was added to give aconcentration of 1M. Then, the hypothalamus was homogenized with aPolytron. After overnight stirring, supernatant was obtained bycentrifugation. Trifluoroacetic acid (TFA) was added to the supernatantto give a concentration of 0.05%, and the supernatant was applied to aC18 column (Prep C18 125 Å; Waters). The peptide bound to the column waseluted in a step-wise manner with 0.05% TFA-containing 10%, 30% and 50%acetonitrile solution. 30% acetonitrile fraction was diluted with twovolumes of 20 mM ammonium acetate (pH 4.7) and applied to an ionexchange column HiPrep C4-Sepharose FF (Pharmacia). The peptide bound tothe ion exchange column was eluted in a stepwise manner with 0.1, 0.2,0.5 and 1.0 M NaCl in 10% acetonitrile-containing 20 mM ammonium acetate(pH 4.7). Three volumes of cold acetone was added to 0.2 M NaCl fractionwhich contained the highest RFRP-3-like immune activity, followed bycentrifugation to remove the precipitate. The resultant supernatant wasconcentrated in an evaporator. TFA was added to the concentratedsupernatant to give a concentration of 0.1%, and then the supernatantwas applied to a reversed-phase HPLC column RESOURCE RPC (Pharmacia) forfurther separation. Elution from the RESOURCE RPC column was performedwith the density gradient of 10-30% acetonitrile. The major RFRP-3-likeactivity was eluted with approximately 23% acetonitrile. This activefraction was separated in a cation exchange column TSK gel CM-SW (Tosoh)using the density gradient of 0.2-0.6 M NaCl in 10%acetonitrile-containing 20 mM ammonium acetate (pH 4.7). The majorRFRP-3-like activity was eluted with approximately 0.36 M NaCl. TFA wasadded to the RFRP-3-like activity-containing CM-2SW column fraction togive a concentration of 0.1%. Then, this fraction was purified finallyin a reversed-phase column μRPC C2/C18 SC2.1/10 using the densitygradient of 18-23% acetonitrile to thereby obtain a single peak which isconsistent with the RFRP-3-like immune activity (FIG. 17).

Example B4 Analysis of the N-Terminal Amino Acid Sequence of the FinallyPurified Product and Determination of its Molecular Weight by MassSpectrometry

[0835] When N-terminal amino acids of the finally purified productobtained in Example B3 were analyzed with a protein sequencer (Model491cLC; Applied Biosystems), the amino acid sequence shown in FIG. 18was obtained.

[0836] When the molecular weight of the finally purified product wasdetermined with ESI-MS (LCQ; Thermoquest), a value 3302 was obtained(FIG. 19). The MS/MS spectrum measured with a pentavalentmolecule-related ion (m/z 661) as a precursor ion was assigned to thestructure of a peptide consisting of the 28 residues starting from Ala104 of RFRP precursor (FIG. 20). From these results, it was found thatthe finally purified product from bovine hypothalamus is a peptideconsisting of the 28 amino acids from Ala 104 to Phe 131 of bovine RFRPprecursor.

Example B5 Preparation of Anti-Rat RFRP-3 Polyclonal Antibody

[0837] A polyclonal antibody was prepared against a peptideNMEAGTMSGFPSLPQRF-Cys as an antigen; this peptide is rat-type RFRP-3consisting of 17 amino acids to which one Cys residue is added at theC-terminus. The antigen peptide (0.6 mg) was conjugated with bovineserum albumin (BSA) using maleimide. The resultant conjugate (500 μg)was subcutaneously injected into rabbits four times. Then, blood wastaken from them to obtain antisera. Two volumes of 3 M ammonium sulfatewas added to 10 ml of each anti-serum, followed by preparation of IgGfraction. This fraction was fed to an antigen column (Sulfo-link column(Pierce) to which the antigen is bound) for affinity purification. Thebound fraction was obtained as anti-rat RFRP-3 polyclonal antibody.

Example B6 Construction of Competitive EIA

[0838] First, the peptide used as the antigen was conjugated with horseradish peroxidase (HRP) using maleimide to thereby prepare a conjugateHRP-rat RFRP-3 (N). Then, using this conjugate HRP-rat RFRP-3 (N) andthe anti-rat RFRP-3 polyclonal antibody obtained in Example B5, acompetitive EIA was constructed.

[0839] Briefly, 50 μl of the anti-rat RFRP-3 polyclonal antibody dilutedwith a buffer (phosphate buffered saline (PBS) containing 2 mM EDTA,0.4% BSA, 0.1 M NaCl and 0.1% micro-O-protect) was added to each well of96-well plates coated with anti-rabbit IgG (Cappel) at 1.5 μg/well andblocked with Block ACE (Dainippon Pharmaceutical). A 50 μl sampledissolved in the same buffer was also added to each well. After a 16-hrincubation at 4° C., 50 μl of HRP-rat RFRP-3 (N) diluted with the bufferwas added to each well. After 2-hr incubation at room temperature, theplates were washed with 0.1% Tween 20 (Sigma)-containing PBS. Then, theactivity of HRP bound to each well was detected by a color reactionusing TMB microwell peroxidase system, followed by measurement of theabsorbance at 450 nm. Changes in absorbance when RFRP-3-related peptideswere added are shown in FIG. 21.

Example B7 Preparation of Human-Type RFRP-3(28)

[0840] Ala-Thr-Ala-Asn-Leu-Pro-Leu-Arg-Ser-Gly-Arg-Asn-Met-Glu-Val-Ser-Leu-Val-Arg-Arg-Val-Pro-Asn-Leu- Pro-Gln-Arg-Phe-NH₂(SEQ ID NO: 63)

[0841] Commercial p-methyl BHA resin (Applied Biosystems) (0.5 mmole)was placed in a reactor in a peptide synthesizer (Applied BiosystemsModel 430A), and swollen with DCM. Then, the first amino acid Boc-Phewas activated by the HOBt/DCC method and introduced into the p-methylBHA resin. Subsequently, the resin was treated with 50% TFA/DCM toremove the Boc group to thereby liberate the amino group, and thenneutralized with DIEA. The subsequent amino acid Boc-Arg(Tos) wascondensed with this amino group by the HOBt/DCC method. The presence orabsence of unreacted amino groups was examined by a ninhydrin test. Ifunreacted amino groups remained, the condensation was repeated. When thecompletion of the reaction was confirmed, Boc-Gln, Boc-Pro, Boc-Leu,Boc-Asn, Boc-Val, Boc-Arg(Tos), Boc-Ser(Bzl), Boc-Glu(OcHex), Boc-Met,Boc-Asn, Boc-Gly and Boc-Thr(Bzl) were condensed in the same manneraccording to the order of the sequence of human-type RFRP-3(28) tothereby obtainBoc-Ala-Thr(Bzl)-Ala-Asn-Leu-Pro-Leu-Arg(Tos)-Ser(Bzl)-Gly-Arg(Tos)-Asn-Met-Glu(OcHex)-Val-Ser(Bzl)-Leu-Val-Arg(Tos)-Arg(Tos)-Val-Pro-Asn-Leu-Pro-Gln-Arg(Tos)-Phe-pMBHA resin.

[0842] In a hydrogen fluoride reactor made of Teflon, 0.3 g of thisresin was reacted with 3.1 g of p-cresol and 15 ml of hydrogen fluorideat 0° C. for 60 min. The hydrogen was distilled off under reducedpressure. The residue was diluted with 100 ml of diethyl ether, stirred,filtered through a glass filter, and the fraction on the filter wasdried. This fraction was suspended in 50 ml of 50% aqueous solution ofacetic acid and stirred to extract the peptide. After the extraction,the resin was separated and the peptide was concentrated under reducedpressure to about 5 ml. This peptide was applied to a Sephadex G-25column (2×90 cm). Development was carried out with 50% aqueous aceticacid solution, and major fractions were collected and lyophilized.Subsequently, this crude peptide was dissolved in 1.5 ml of 5%thioglycollic acid/50% acetic acid and retained at 50° C. for 12 hr toreduce the Met-oxidant peptide. Then, the peptide was applied to areversed phase column packed with LiChroprep RP-18 (Merck) andrepeatedly purified by gradient elution using 0.1% TFA/H₂O and 0.1%TFA-containing 33% acetonitrile/H₂O. Major fractions were pooled andlyophilized to obtain 30 mg of white powder.

[0843] (M+H)⁺ value by mass spectrometry: 3190.9

[0844] HPLC elution time: 15.5 min

[0845] Column conditions:

[0846] Column: Wakosil II5C18HG (4.6×100 mm)

[0847] Eluent:

[0848] A (0.1% TFA-containing 5% acetonitrile/H₂O)

[0849] B (0.1% TFA-containing 55% acetonitrile/H₂O)

[0850] Linear gradient elution from A to B (25 min.)

[0851] Flow rate: 1.0 ml/min

Example B8 Preparation of Human-Type RFRP-3(31)

[0852] Ser-Ala-Gly-Ala-Thr-Ala-Asn-Leu-Pro-Leu-Arg-Ser-Gly-Arg-Asn-Met-Glu-Val-Ser-Leu-Val-Arg-Arg-Val-Pro-Asn-Leu-Pro-Gln-Arg-Phe-NH₂ (SEQ ID NO: 65)

[0853] Boc-Gly, Boc-Ala and Boc-Ser(Bzl) were further condensed insuccession in the same manner with theBoc-Ala-Thr(Bzl)-Ala-Asn-Leu-Pro-Leu-Arg(Tos)-Ser(Bzl)-Gly-Arg(Tos)-Asn-Met-Glu(OcHex)-Val-Ser(Bzl)-Leu-Val-Arg(Tos)-Arg(Tos)-Val-Pro-Asn-Leu-Pro-Gln-Arg(Tos)-Phe-pMBHAresin prepared in Example B7 to thereby obtainBoc-Ser(Bzl)-Ala-Gly-Ala-Thr(Bzl)-Ala-Asn-Leu-Pro-Leu-Arg(Tos)-Ser(Bzl)-Gly-Arg(Tos)-Asn-Met-Glu(OcHex)-Val-Ser(Bzl)-Leu-Val-Arg(Tos)-Arg(Tos)-Val-Pro-Asn-Leu-Pro-Gln-Arg(Tos)-Phe-pMBHAresin.

[0854] This resin was treated with hydrogen fluoride in the same manneras in Example B7. The resultant crude peptide was purified in the samemanner to thereby obtain 25 mg of white powder.

[0855] (M+H)⁺ value by mass spectrometry: 3405.9

[0856] HPLC elution time: 15.7 min

[0857] Column conditions:

[0858] Column: Wakosil II5C18HG (4.6×100 mm)

[0859] Eluent:

[0860] A (0.1% TFA-containing 5% acetonitrile/H₂O)

[0861] B (0.1% TFA-containing 55% acetonitrile/H₂O)

[0862] Linear gradient elution from A to B (25 min.)

[0863] Flow rate: 1.0 ml/min

Example B9 Preparation of Leu-Pro-Gln-Arg-Phe-NH₂ (SEQ ID NO: 69):hRFRP-3(5)

[0864] In the course of preparation of the resin of Example B7, a partof Boc-Leu-Pro-Gln-Arg(Tos)-Phe-pMBHA resin was taken out, treated withhydrogen fluoride, purified in a Sephadex G-25 column (2×90 cm) andfurther purified in a reversed-phase column packed with LiChroprep™RP-18 (Merck) in the same manner as in Examples B7 and B8, to therebyobtain Leu-Pro-Gln-Arg-Phe-NH₂.

Example B10 Preparation of Asn-Leu-Pro-Gln-Arg-Phe-NH₂ (SEQ ID NO: 70):hRFRP-3(6)

[0865] In the course of preparation of the resin of Example B7, a partof Boc-Asn-Leu-Pro-Gln-Arg(Tos)-Phe-pMBHA resin was taken out, treatedwith hydrogen fluoride, purified in a Sephadex G-25 column (2×90 cm) andfurther purified in a reversed-phase column packed with LiChroprep™RP-18 (Merck) in the same manner as in Examples B7 and B8, to therebyobtain Asn-Leu-Pro-Gln-Arg-Phe-NH₂.

Example B11 Preparation of Pro-Asn-Leu-Pro-Gln-Arg-Phe-NH₂ (SEQ ID NO:71): hRFRP-3(7)

[0866] In the course of preparation of the resin of Example B7, a partof Boc-Pro-Asn-Leu-Pro-Gln-Arg(Tos)-Phe-pMBHA resin was taken out,treated with hydrogen fluoride, purified in a Sephadex G-25 column (2×90cm) and further purified in a reversed-phase column packed withLiChroprep™ RP-18 (Merck) in the same manner as in Examples B7 and B8,to thereby obtain Pro-Asn-Leu-Pro-Gln-Arg-Phe-NH₂.

Example B12 Preparation of ¹²⁵I-Labeled hRFRP-3(8)

[0867] Twenty microliters of Tyr-hRFRP-3(8) (0.1 M), 10 μl of distilledwater, 20 μl of lactoperoxidase (Sigma; diluted to 10 μg/ml with 0.1 MHEPES-NaOH pH 7.0), 10 μl of Iodine-125 (Amersham; IMS-30, 74 MBq) and20 μl of 0.005% hydrogen peroxide (Wako Purechemical Industries) weremixed and left stationary at room temperature for 10 min. Then, 600 μlof 0.1% TFA was added thereto. The resultant mixture was separated byreversed-phase HPLC and the peak of the labeled peptide was recovered.This fraction was immediately stored on ice, and an equal volume ofassay buffer [50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.1% bovine serumalbumin (Sigma), 0.5 mM PMSF (Wako Purechemical Industries), 20 μg/mlleupeptin (Peptide Institute, Inc.), 0.1 μg/ml pepstatin A (PeptideInstitute, Inc.), 4 μg/ml E64 (Peptide Institute, Inc.) and 10 mM MgCl₂]was added thereto. An aliquot of this mixture was taken and subjected tomeasurement of radioactivity with a γ counter. The remaining sample wasdivided into 100 μl aliquots and stored frozen.

Example B13 Preparation of the Membrane Fraction from Human-TypeOT7T022-Expressing CHO Cells

[0868] A flask used for culturing human-type OT7T022-expressing CHOcells was washed with 5 mM EDTA/PBS. The cells were peeled off using 5mM EDTA/PBS, centrifuged, and recovered. Then, the cells were suspendedin 25 ml of membrane fraction preparation buffer [50 mM Tris-HCl, pH7.5, 5 mM EDTA, 0.1% bovine serum albumin (Sigma), 0.5 mM PMSF (WakoPurechemical Industries), 20 μl g/ml leupeptin (Peptide Institute,Inc.), 0.1 μg/ml pepstatin A (Peptide Institute, Inc.) and 4 μg/ml E-64(Peptide Institute, Inc.)] and homogenized on ice using a Polytron(12,000 rpm; 15 sec X 3 times). The homogenized cells were centrifugedin a high-speed refrigerated microcentrifuge at 4° C., at 1,000 g for 10min to recover the supernatant. To the precipitate, 25 ml of themembrane fraction preparation buffer was added and centrifuged in thesame manner to recover the supernatant. These supernatants werecollected together, passed through a cell strainer and dispensed intocentrifugal tubes, followed by centrifugation at 4° C. at 100,000 g for1 hr. The resultant pellet was recovered, suspended in a small amount ofthe membrane fraction preparation buffer and suspended with a Teflonhomogenizer. Subsequently, an aliquot of this sample was used formeasuring the amount of protein, and the remaining sample was dispensedinto small aliquots and stored at −80° C.

Example B14 Binding Inhibition Experiment

[0869] An assay buffer [50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.1% bovineserum albumin (Sigma), 0.5 mM PMSF (Wako Purechemical Industries), 20μg/ml leupeptin (Peptide Institute, Inc.), 0.1 μg/ml pepstatin A(Peptide Institute, Inc.), 4 μg/ml E64 (Peptide Institute, Inc.) and 10mM MgCl₂] was prepared. With this buffer, the membrane fraction fromhuman-type OT7T022-expressing CHO cells was diluted to give aconcentration of 1 μg/25 μl. With respect to the peptides shown in Table1, 10-2 M or 10⁻³M stock solution was diluted with the assay buffer togive 2-fold concentrations of the assay concentrations (10⁻⁵M, 10⁻⁶M,10⁻⁷M, 10⁻⁸M, 10⁻⁹M, 10⁻¹⁰M, 10⁻¹¹M). In order to measure non-specificbinding, 20 μM Tyr-hRFRP-3(8) was prepared. Sample solutions prepared,NSB and the assay buffer were dispensed into polypropylene 96-wellplates in quadruplicate as total 50 μl/well. With respect to the labeledpeptide, the ¹²⁵I-Tyr-hRFRP-3(8) prepared was diluted to 400 pM withmeasuring buffer, and 25 u 1 of this dilution was added to each well andagitated with a plate mixer. Twenty-five microliter of the membranefraction solution from human-type OT7T022-expressing CHO cells was addedthereto, agitated with a plate mixer and incubated at room temperaturefor 1.5 hr. Using a cell harvester for 96-well plates, the contents ofeach well were adsorbed onto a filter unit (GF/C, treated withpolyethylene imine) pre-moisturized with washing buffer (50 mM Tris-HCl,pH 7.5). After washing 5 times with the washing buffer, the adsorbatewas dried thoroughly. The lower part of the filter unit was sealed.Then, 50 μl of liquid scintillator was added to each well and the upperpart of the filter unit was sealed. Radioactivity was measured with aTop Count (Packard), and data were analyzed in triplicate. IC₅₀ valuesare shown in Table 1.

Example B15 cAMP Production Inhibition Experiment

[0870] Human-type OT7T022-expressing CHO cells were subcultured to24-well plates at 3×10⁵ cells/well and cultured for one day at 37° C.under 5% CO₂ 95% air. An assay buffer was prepared by adding to Hanks'Balanced Salt Solution (Gibco) 0.05% bovine serum albumin and 200 μM3-isobutyl-1-methylxanthine (Sigma). The plates cultured overnight werewashed twice with 400 μl of the assay buffer. Then, the medium wasexchanged for 400 μl of the assay buffer, and the cells were cultured at37° C., for 30 min under 100% air. A sample dilution buffer was preparedby adding 1 μM forskolin to the assay buffer. Using this sample dilutionbuffer, stock solutions (10⁻² M or 10⁻³ M) of the peptides shown inTable 1 were diluted to prepare 10⁻⁶M, 10⁻⁷M, 10⁻⁸M, 10⁻⁹M and 10⁻¹⁰Msample solutions. The plates cultured for 30 min with the assay bufferwere taken out, washed with the assay buffer once, and 500 μl of asample solution was added thereto. For each sample, measurement wasperformed in triplicate. Further, the equal volume of the assay bufferwas added for measuring the basal level; and the equal volume of thesample dilution buffer was added for measuring the maximum level. Plateswere cultured at 37° C. for 30 min under 100% air. Then, the amount ofintracellular cAMP was determined with Biotaak™ cAMP enzyme immunoassay(EIA) system (Amersham Pharmacia Biotech) according to the protocolattached to this system. The difference between the maximum cAMP leveland cAMP level when each sample was added was calculated, and thenpercentage against the amount of cAMP production promoted by forskolinwas calculated. This percentage was taken as inhibition ratio againstcAMP production. EC₅₀ values of individual samples are shown in Table 1.TABLE 1 Binding inhibition cAMP production inhibition IC₅₀ (nM) EC₅₀(nM) hRFRP-3 (31) 1.3 4.8 hRFRP-3 (28) 1.1 5.1 hRFRP-3 (8) 1.2 4.1hRFRP-3 (7) 0.62 3.2 hRFRP-3 (6) 2.6 37 hRFRP-3 (5) 2.1 38 hRFRP-3 (28)1.6 4.1

INDUSTRIAL APPLICABILITY

[0871] The RFRP-3 of the invention has both promoting and inhibitingeffects upon prolactin secretion. This means that the RFRP-3 of theinvention may be used as prophylactic and therapeutic agents for variousdiseases associated with prolactin secretion insufficiency based on itsprolactin secretion-promoting effect. On the other hand, since theRFRP-3 of the invention has strong affinity with its receptor protein,when administered at a high dose the RFRP-3 of the invention causesdesensitization against prolactin secretion and, as a result, has aneffect in inhibiting prolactin secretion. In such cases, the RFRP-3 ofthe invention may be used as prophylactic and therapeutic agents forvarious diseases associated with excessive prolactin secretion.

1 72 1 180 PRT Human 1 Met Glu Ile Ile Ser Ser Lys Leu Phe Ile Leu LeuThr Leu Ala Thr 1 5 10 15 Ser Ser Leu Leu Thr Ser Asn Ile Phe Cys AlaAsp Glu Leu Val Met 20 25 30 Ser Asn Leu His Ser Lys Glu Asn Tyr Asp LysTyr Ser Glu Pro Arg 35 40 45 Gly Tyr Pro Lys Gly Glu Arg Ser Leu Asn PheGlu Glu Leu Lys Asp 50 55 60 Trp Gly Pro Lys Asn Val Ile Lys Met Ser ThrPro Ala Val Asn Lys 65 70 75 80 Met Pro His Ser Phe Ala Asn Leu Pro LeuArg Phe Gly Arg Asn Val 85 90 95 Gln Glu Glu Arg Ser Ala Gly Ala Thr AlaAsn Leu Pro Leu Arg Ser 100 105 110 Gly Arg Asn Met Glu Val Ser Leu ValArg Arg Val Pro Asn Leu Pro 115 120 125 Gln Arg Phe Gly Arg Thr Thr ThrAla Lys Ser Val Cys Arg Met Leu 130 135 140 Ser Asp Leu Cys Gln Gly SerMet His Ser Pro Cys Ala Asn Asp Leu 145 150 155 160 Phe Tyr Ser Met ThrCys Gln His Gln Glu Ile Gln Asn Pro Asp Gln 165 170 175 Lys Gln Ser Arg180 2 540 DNA Human 2 atggaaatta tttcatcaaa actattcatt ttattgactttagccacttc aagcttgtta 60 acatcaaaca ttttttgtgc agatgaatta gtgatgtccaatcttcacag caaagaaaat 120 tatgacaaat attctgagcc tagaggatac ccaaaaggggaaagaagcct caattttgag 180 gaattaaaag attggggacc aaaaaatgtt attaagatgagtacacctgc agtcaataaa 240 atgccacact ccttcgccaa cttgccattg agatttgggaggaacgttca agaagaaaga 300 agtgctggag caacagccaa cctgcctctg agatctggaagaaatatgga ggtgagcctc 360 gtgagacgtg ttcctaacct gccccaaagg tttgggagaacaacaacagc caaaagtgtc 420 tgcaggatgc tgagtgattt gtgtcaagga tccatgcattcaccatgtgc caatgactta 480 ttttactcca tgacctgcca gcaccaagaa atccagaatcccgatcaaaa acagtcaagg 540 3 27 DNA Artificial Sequence primer 3gggctgcaca tagagactta attttag 27 4 27 DNA Artificial Sequence primer 4ctagaccacc tctatataac tgcccat 27 5 30 DNA Artificial Sequence primer 5gcacatagag acttaatttt agatttagac 30 6 27 DNA Artificial Sequence primer6 catgcacttt gactggtttc caggtat 27 7 27 DNA Artificial Sequence primer 7cagctttagg gacaggctcc aggtttc 27 8 196 PRT Human 8 Met Glu Ile Ile SerSer Lys Leu Phe Ile Leu Leu Thr Leu Ala Thr 1 5 10 15 Ser Ser Leu LeuThr Ser Asn Ile Phe Cys Ala Asp Glu Leu Val Met 20 25 30 Ser Asn Leu HisSer Lys Glu Asn Tyr Asp Lys Tyr Ser Glu Pro Arg 35 40 45 Gly Tyr Pro LysGly Glu Arg Ser Leu Asn Phe Glu Glu Leu Lys Asp 50 55 60 Trp Gly Pro LysAsn Val Ile Lys Met Ser Thr Pro Ala Val Asn Lys 65 70 75 80 Met Pro HisSer Phe Ala Asn Leu Pro Leu Arg Phe Gly Arg Asn Val 85 90 95 Gln Glu GluArg Ser Ala Gly Ala Thr Ala Asn Leu Pro Leu Arg Ser 100 105 110 Gly ArgAsn Met Glu Val Ser Leu Val Arg Arg Val Pro Asn Leu Pro 115 120 125 GlnArg Phe Gly Arg Thr Thr Thr Ala Lys Ser Val Cys Arg Met Leu 130 135 140Ser Asp Leu Cys Gln Gly Ser Met His Ser Pro Cys Ala Asn Asp Leu 145 150155 160 Phe Tyr Ser Met Thr Cys Gln His Gln Glu Ile Gln Asn Pro Asp Gln165 170 175 Lys Gln Ser Arg Arg Leu Leu Phe Lys Lys Ile Asp Asp Ala GluLeu 180 185 190 Lys Gln Glu Lys 195 9 588 DNA Human 9 atggaaattatttcatcaaa actattcatt ttattgactt tagccacttc aagcttgtta 60 acatcaaacattttttgtgc agatgaatta gtgatgtcca atcttcacag caaagaaaat 120 tatgacaaatattctgagcc tagaggatac ccaaaagggg aaagaagcct caattttgag 180 gaattaaaagattggggacc aaaaaatgtt attaagatga gtacacctgc agtcaataaa 240 atgccacactccttcgccaa cttgccattg agatttggga ggaacgttca agaagaaaga 300 agtgctggagcaacagccaa cctgcctctg agatctggaa gaaatatgga ggtgagcctc 360 gtgagacgtgttcctaacct gccccaaagg tttgggagaa caacaacagc caaaagtgtc 420 tgcaggatgctgagtgattt gtgtcaagga tccatgcatt caccatgtgc caatgactta 480 ttttactccatgacctgcca gcaccaagaa atccagaatc ccgatcaaaa acagtcaagg 540 agactgctattcaagaaaat agatgatgca gaattgaaac aagaaaaa 588 10 27 DNA ArtificialSequence primer 10 gcctagagga gatctaggct gggagga 27 11 27 DNA ArtificialSequence primer 11 gggaggaaca tggaagaaga aaggagc 27 12 27 DNA ArtificialSequence primer 12 gatggtgaat gcatggactg ctggagc 27 13 27 DNA ArtificialSequence primer 13 ttcctcccaa atctcagtgg caggttg 27 14 196 PRT Bovine 14Met Glu Ile Ile Ser Leu Lys Arg Phe Ile Leu Leu Met Leu Ala Thr 1 5 1015 Ser Ser Leu Leu Thr Ser Asn Ile Phe Cys Thr Asp Glu Ser Arg Met 20 2530 Pro Asn Leu Tyr Ser Lys Lys Asn Tyr Asp Lys Tyr Ser Glu Pro Arg 35 4045 Gly Asp Leu Gly Trp Glu Lys Glu Arg Ser Leu Thr Phe Glu Glu Val 50 5560 Lys Asp Trp Ala Pro Lys Ile Lys Met Asn Lys Pro Val Val Asn Lys 65 7075 80 Met Pro Pro Ser Ala Ala Asn Leu Pro Leu Arg Phe Gly Arg Asn Met 8590 95 Glu Glu Glu Arg Ser Thr Arg Ala Met Ala His Leu Pro Leu Arg Leu100 105 110 Gly Lys Asn Arg Glu Asp Ser Leu Ser Arg Trp Val Pro Asn LeuPro 115 120 125 Gln Arg Phe Gly Arg Thr Thr Thr Ala Lys Ser Ile Thr LysThr Leu 130 135 140 Ser Asn Leu Leu Gln Gln Ser Met His Ser Pro Ser ThrAsn Gly Leu 145 150 155 160 Leu Tyr Ser Met Ala Cys Gln Pro Gln Glu IleGln Asn Pro Gly Gln 165 170 175 Lys Asn Leu Arg Arg Arg Gly Phe Gln LysIle Asp Asp Ala Glu Leu 180 185 190 Lys Gln Glu Lys 195 15 588 DNABovine 15 atggaaatta tttcattaaa acgattcatt ttattgatgt tagccacttcaagcttgtta 60 acatcaaaca tcttctgcac agacgaatca aggatgccca atctttacagcaaaaagaat 120 tatgacaaat attccgagcc tagaggagat ctaggctggg agaaagaaagaagtcttact 180 tttgaagaag taaaagattg ggctccaaaa attaagatga ataaacctgtagtcaacaaa 240 atgccacctt ctgcagccaa cctgccactg agatttggga ggaacatggaagaagaaagg 300 agcactaggg cgatggccca cctgcctctg agactcggaa aaaatagagaggacagcctc 360 tccagatggg tcccaaatct gccccagagg tttggaagaa caacaacagccaaaagcatt 420 accaagaccc tgagtaattt gctccagcag tccatgcatt caccatctaccaatgggcta 480 ctctactcca tggcctgcca gccccaagaa atccagaatc ctggtcaaaagaacctaagg 540 agacggggat tccagaaaat agatgatgca gaattgaaac aagaaaaa 58816 27 DNA Artificial Sequence primer 16 ccctggggct tcttctgtct tctatgt 2717 26 DNA Artificial Sequence primer 17 agcgattcat tttattgact ttagca 2618 203 PRT Rat 18 Met Glu Ile Ile Ser Ser Lys Arg Phe Ile Leu Leu ThrLeu Ala Thr 1 5 10 15 Ser Ser Phe Leu Thr Ser Asn Thr Leu Cys Ser AspGlu Leu Met Met 20 25 30 Pro His Phe His Ser Lys Glu Gly Tyr Gly Lys TyrTyr Gln Leu Arg 35 40 45 Gly Ile Pro Lys Gly Val Lys Glu Arg Ser Val ThrPhe Gln Glu Leu 50 55 60 Lys Asp Trp Gly Ala Lys Lys Asp Ile Lys Met SerPro Ala Pro Ala 65 70 75 80 Asn Lys Val Pro His Ser Ala Ala Asn Leu ProLeu Arg Phe Gly Arg 85 90 95 Asn Ile Glu Asp Arg Arg Ser Pro Arg Ala ArgAla Asn Met Glu Ala 100 105 110 Gly Thr Met Ser His Phe Pro Ser Leu ProGln Arg Phe Gly Arg Thr 115 120 125 Thr Ala Arg Arg Ile Thr Lys Thr LeuAla Gly Leu Pro Gln Lys Ser 130 135 140 Leu His Ser Leu Ala Ser Ser GluSer Leu Tyr Ala Met Thr Arg Gln 145 150 155 160 His Gln Glu Ile Gln SerPro Gly Gln Glu Gln Pro Arg Lys Arg Val 165 170 175 Phe Thr Glu Thr AspAsp Ala Glu Arg Lys Gln Glu Lys Ile Gly Asn 180 185 190 Leu Gln Pro ValLeu Gln Gly Ala Met Lys Leu 195 200 19 609 DNA Rat 19 atggaaattatttcatcaaa gcgattcatt ttattgactt tagcaacttc aagcttctta 60 acttcaaacaccctttgttc agatgaatta atgatgcccc attttcacag caaagaaggt 120 tatggaaaatattaccagct gagaggaatc ccaaaagggg taaaggaaag aagtgtcact 180 tttcaagaactcaaagattg gggggcaaag aaagatatta agatgagtcc agcccctgcc 240 aacaaagtgccccactcagc agccaacctt cccctgaggt ttgggaggaa catagaagac 300 agaagaagccccagggcacg ggccaacatg gaggcaggga ccatgagcca ttttcccagc 360 ctgccccaaaggtttgggag aacaacagcc agacgcatca ccaagacact ggctggtttg 420 ccccagaaatccctgcactc cctggcctcc agtgaatcgc tctatgccat gacccgccag 480 catcaagaaattcagagtcc tggtcaagag caacctagga aacgggtgtt cacggaaaca 540 gatgatgcagaaaggaaaca agaaaaaata ggaaacctcc agccagtcct tcaaggggct 600 atgaagctg 60920 12 DNA Artificial Sequence misc_feature (3)..(3) a, c, g or t 20mgnttyggna ar 12 21 12 DNA Artificial Sequence misc_feature (3)..(3) a,c, g or t 21 mgnttyggnm gn 12 22 12 DNA Artificial Sequence misc_feature(3)..(3) a, c, g or t 22 mgnwsnggna ar 12 23 12 DNA Artificial Sequencemisc_feature (3)..(3) a, c, g or t 23 mgnwsnggnm gn 12 24 12 DNAArtificial Sequence misc_feature (3)..(3) a, c, g or t 24 mgnytnggna ar12 25 12 DNA Artificial Sequence misc_feature (3)..(3) a, c, g or t 25mgnytnggnm gn 12 26 30 DNA Artificial Sequence primer 26 gacttaattttagatttaga caaaatggaa 30 27 25 DNA Artificial Sequence primer 27ttctcccaaa cctttggggc aggtt 25 28 28 DNA Artificial Sequence primer 28acagcaaaga aggtgacgga aaatactc 28 29 28 DNA Artificial Sequence primer29 atagatgaga aaagaagccc cgcagcac 28 30 28 DNA Artificial Sequenceprimer 30 gtgctgcggg gcttcttttc tcatctat 28 31 21 DNA ArtificialSequence primer 31 tttagactta gacgaaatgg a 21 32 21 DNA ArtificialSequence primer 32 gctccgtagc ctcttgaagt c 21 33 188 PRT Mouse 33 MetGlu Ile Ile Ser Leu Lys Arg Phe Ile Leu Leu Thr Val Ala Thr 1 5 10 15Ser Ser Phe Leu Thr Ser Asn Thr Phe Cys Thr Asp Glu Phe Met Met 20 25 30Pro His Phe His Ser Lys Glu Gly Asp Gly Lys Tyr Ser Gln Leu Arg 35 40 45Gly Ile Pro Lys Gly Glu Lys Glu Arg Ser Val Ser Phe Gln Glu Leu 50 55 60Lys Asp Trp Gly Ala Lys Asn Val Ile Lys Met Ser Pro Ala Pro Ala 65 70 7580 Asn Lys Val Pro His Ser Ala Ala Asn Leu Pro Leu Arg Phe Gly Arg 85 9095 Thr Ile Asp Glu Lys Arg Ser Pro Ala Ala Arg Val Asn Met Glu Ala 100105 110 Gly Thr Arg Ser His Phe Pro Ser Leu Pro Gln Arg Phe Gly Arg Thr115 120 125 Thr Ala Arg Ser Pro Lys Thr Pro Ala Asp Leu Pro Gln Lys ProLeu 130 135 140 His Ser Leu Gly Ser Ser Glu Leu Leu Tyr Val Met Ile CysGln His 145 150 155 160 Gln Glu Ile Gln Ser Pro Gly Gly Lys Arg Thr ArgArg Gly Ala Phe 165 170 175 Val Glu Thr Asp Asp Ala Glu Arg Lys Pro GluLys 180 185 34 564 DNA Mouse 34 atggaaatta tttcattaaa acgattcattttattgactg tggcaacttc aagcttctta 60 acatcaaaca ccttctgtac agatgagttcatgatgcctc attttcacag caaagaaggt 120 gacggaaaat actcccagct gagaggaatcccaaaagggg aaaaggaaag aagtgtcagt 180 tttcaagaac taaaagattg gggggcaaagaatgttatta agatgagtcc agcccctgcc 240 aacaaagtgc cccactcagc agccaacctgcccctgagat ttggaaggac catagatgag 300 aaaagaagcc ccgcagcacg ggtcaacatggaggcaggga ccaggagcca tttccccagc 360 ctgccccaaa ggtttgggag aacaacagccagaagcccca agacacccgc tgatttgcca 420 cagaaacccc tgcactcact gggctccagcgagttgctct acgtcatgat ctgccagcac 480 caagaaattc agagtcctgg tggaaagcgaacgaggagag gagcgtttgt ggaaacagat 540 gatgcagaaa ggaaaccaga aaaa 564 3527 DNA Artificial Sequence primer 35 agtcgacagt atggaggcgg agccctc 27 3629 DNA Artificial Sequence primer 36 gactagttca aatgttccag gccgggatg 2937 432 PRT Rat 37 Met Glu Ala Glu Pro Ser Gln Pro Pro Asn Gly Ser TrpPro Leu Gly 5 10 15 Gln Asn Gly Ser Asp Val Glu Thr Ser Met Ala Thr SerLeu Thr Phe 20 25 30 Ser Ser Tyr Tyr Gln His Ser Ser Pro Val Ala Ala MetPhe Ile Ala 35 40 45 Ala Tyr Val Leu Ile Phe Leu Leu Cys Met Val Gly AsnThr Leu Val 50 55 60 Cys Phe Ile Val Leu Lys Asn Arg His Met Arg Thr ValThr Asn Met 65 70 75 80 Phe Ile Leu Asn Leu Ala Val Ser Asp Leu Leu ValGly Ile Phe Cys 85 90 95 Met Pro Thr Thr Leu Val Asp Asn Leu Ile Thr GlyTrp Pro Phe Asp 100 105 110 Asn Ala Thr Cys Lys Met Ser Gly Leu Val GlnGly Met Ser Val Ser 115 120 125 Ala Ser Val Phe Thr Leu Val Ala Ile AlaVal Glu Arg Phe Arg Cys 130 135 140 Ile Val His Pro Phe Arg Glu Lys LeuThr Leu Arg Lys Ala Leu Phe 145 150 155 160 Thr Ile Ala Val Ile Trp AlaLeu Ala Leu Leu Ile Met Cys Pro Ser 165 170 175 Ala Val Thr Leu Thr ValThr Arg Glu Glu His His Phe Met Leu Asp 180 185 190 Ala Arg Asn Arg SerTyr Pro Leu Tyr Ser Cys Trp Glu Ala Trp Pro 195 200 205 Glu Lys Gly MetArg Lys Val Tyr Thr Ala Val Leu Phe Ala His Ile 210 215 220 Tyr Leu ValPro Leu Ala Leu Ile Val Val Met Tyr Val Arg Ile Ala 225 230 235 240 ArgLys Leu Cys Gln Ala Pro Gly Pro Ala Arg Asp Thr Glu Glu Ala 245 250 255Val Ala Glu Gly Gly Arg Thr Ser Arg Arg Arg Ala Arg Val Val His 260 265270 Met Leu Val Met Val Ala Leu Phe Phe Thr Leu Ser Trp Leu Pro Leu 275280 285 Trp Val Leu Leu Leu Leu Ile Asp Tyr Gly Glu Leu Ser Glu Leu Gln290 295 300 Leu His Leu Leu Ser Val Tyr Ala Phe Pro Leu Ala His Trp LeuAla 305 310 315 320 Phe Phe His Ser Ser Ala Asn Pro Ile Ile Tyr Gly TyrPhe Asn Glu 325 330 335 Asn Phe Arg Arg Gly Phe Gln Ala Ala Phe Arg AlaGln Leu Cys Trp 340 345 350 Pro Pro Trp Ala Ala His Lys Gln Ala Tyr SerGlu Arg Pro Asn Arg 355 360 365 Leu Leu Arg Arg Arg Val Val Val Asp ValGln Pro Ser Asp Ser Gly 370 375 380 Leu Pro Ser Glu Ser Gly Pro Ser SerGly Val Pro Gly Pro Gly Arg 385 390 395 400 Leu Pro Leu Arg Asn Gly ArgVal Ala His Gln Asp Gly Pro Gly Glu 405 410 415 Gly Pro Gly Cys Asn HisMet Pro Leu Thr Ile Pro Ala Trp Asn Ile 420 425 430 38 1299 DNA Rat 38atggaggcgg agccctccca gcctcccaac ggcagctggc ccctgggtca gaacgggagt 60gatgtggaga ccagcatggc aaccagcctc accttctcct cctactacca acactcctct 120ccggtggcag ccatgttcat cgcggcctac gtgctcatct tcctcctctg catggtgggc 180aacaccctgg tctgcttcat tgtgctcaag aaccggcaca tgcgcactgt caccaacatg 240tttatcctca acctggccgt cagcgacctg ctggtgggca tcttctgcat gcccacaacc 300cttgtggaca accttatcac tggttggcct tttgacaacg ccacatgcaa gatgagcggc 360ttggtgcagg gcatgtccgt gtctgcatcg gttttcacac tggtggccat cgctgtggaa 420aggttccgct gcatcgtgca ccctttccgc gagaagctga cccttcggaa ggcgctgttc 480accatcgcgg tgatctgggc tctggcgctg ctcatcatgt gtccctcggc ggtcactctg 540acagtcaccc gagaggagca tcacttcatg ctggatgctc gtaaccgctc ctacccgctc 600tactcgtgct gggaggcctg gcccgagaag ggcatgcgca aggtctacac cgcggtgctc 660ttcgcgcaca tctacctggt gccgctggcg ctcatcgtag tgatgtacgt gcgcatcgcg 720cgcaagctat gccaggcccc cggtcctgcg cgcgacacgg aggaggcggt ggccgagggt 780ggccgcactt cgcgccgtag ggcccgcgtg gtgcacatgc tggtcatggt ggcgctcttc 840ttcacgttgt cctggctgcc actctgggtg ctgctgctgc tcatcgacta tggggagctg 900agcgagctgc aactgcacct gctgtcggtc tacgccttcc ccttggcaca ctggctggcc 960ttcttccaca gcagcgccaa ccccatcatc tacggctact tcaacgagaa cttccgccgc 1020ggcttccagg ctgccttccg tgcacagctc tgctggcctc cctgggccgc ccacaagcaa 1080gcctactcgg agcggcccaa ccgcctcctg cgcaggcggg tggtggtgga cgtgcaaccc 1140agcgactccg gcctgccatc agagtctggc cccagcagcg gggtcccagg gcctggccgg 1200ctgccactgc gcaatgggcg tgtggcccat caggatggcc cgggggaagg gccaggctgc 1260aaccacatgc ccctcaccat cccggcctgg aacatttga 1299 39 12 PRT ArtificialSequence the C-terminus of the polypeptide is amide (-CONH2) form 39 MetPro His Ser Phe Ala Asn Leu Pro Leu Arg Phe 1 5 10 40 8 PRT ArtificialSequence the C-terminus of the polypeptide is amide (-CONH2) form 40 ValPro Asn Leu Pro Gln Arg Phe 1 5 41 11 PRT Artificial Sequence theC-terminus of the polypeptide is amide (-CONH2) form 41 Ser Ala Gly AlaThr Ala Asn Leu Pro Arg Ser 1 5 10 42 36 DNA Human 42 atgccacactccttcgccaa cttgccattg agattt 36 43 36 DNA Human 43 agtgctggag caacagccaacctgcctctg agatct 36 44 24 DNA Human 44 gttcctaacc tgccccaaag gttt 24 45276 DNA Human 45 atggaaatta tttcatcaaa actattcatt ttattgactt tagccacttcaagcttgtta 60 acatcaaaca ttttttgtgc agatgaatta gtgatgtcca atcttcacagcaaagaaaat 120 tatgacaaat attctgagcc tagaggatac ccaaaagggg aaagaagcctcaattttgag 180 gaattaaaag attggggacc aaaaaatgtt attaagatga gtacacctgcagtcaataaa 240 atgccacact ccttcgccaa cttgccattg agattt 276 46 336 DNAHuman 46 atggaaatta tttcatcaaa actattcatt ttattgactt tagccacttcaagcttgtta 60 acatcaaaca ttttttgtgc agatgaatta gtgatgtcca atcttcacagcaaagaaaat 120 tatgacaaat attctgagcc tagaggatac ccaaaagggg aaagaagcctcaattttgag 180 gaattaaaag attggggacc aaaaaatgtt attaagatga gtacacctgcagtcaataaa 240 atgccacact ccttcgccaa cttgccattg agatttggga ggaacgttcaagaagaaaga 300 agtgctggag caacagccaa cctgcctctg agatct 336 47 393 DNAHuman 47 atggaaatta tttcatcaaa actattcatt ttattgactt tagccacttcaagcttgtta 60 acatcaaaca ttttttgtgc agatgaatta gtgatgtcca atcttcacagcaaagaaaat 120 tatgacaaat attctgagcc tagaggatac ccaaaagggg aaagaagcctcaattttgag 180 gaattaaaag attggggacc aaaaaatgtt attaagatga gtacacctgcagtcaataaa 240 atgccacact ccttcgccaa cttgccattg agatttggga ggaacgttcaagaagaaaga 300 agtgctggag caacagccaa cctgcctctg agatctgga agaaatatggaggtgagcctc 360 gtgagacgtg ttcctaacct gccccaaagg ttt 393 48 27 DNAArtificial Sequence primer 48 ccctggggct tcttctgtct tctatgt 27 49 26 DNAArtificial Sequence primer 49 agcgattcat tttattgact ttagca 26 50 203 PRTRat 50 Met Glu Ile Ile Ser Ser Lys Arg Phe Ile Leu Leu Thr Leu Ala Thr 15 10 15 Ser Ser Phe Leu Thr Ser Asn Thr Leu Cys Ser Asp Glu Leu Met Met20 25 30 Pro His Phe His Ser Lys Glu Gly Tyr Gly Lys Tyr Tyr Gln Leu Arg35 40 45 Gly Ile Pro Lys Gly Val Lys Glu Arg Ser Val Thr Phe Gln Glu Leu50 55 60 Lys Asp Trp Gly Ala Lys Lys Asp Ile Lys Met Ser Pro Ala Pro Ala65 70 75 80 Asn Lys Val Pro His Ser Ala Ala Asn Leu Pro Leu Arg Phe GlyArg 85 90 95 Asn Ile Glu Asp Arg Arg Ser Pro Arg Ala Arg Ala Asn Met GluAla 100 105 110 Gly Thr Met Ser His Phe Pro Ser Leu Pro Gln Arg Phe GlyArg Thr 115 120 125 Thr Ala Arg Arg Ile Thr Lys Thr Leu Ala Gly Leu ProGln Lys Ser 130 135 140 Leu His Ser Leu Ala Ser Ser Glu Leu Leu Tyr AlaMet Thr Arg Gln 145 150 155 160 His Gln Glu Ile Gln Ser Pro Gly Gln GluGln Pro Arg Lys Arg Val 165 170 175 Phe Thr Glu Thr Asp Asp Ala Glu ArgLys Gln Glu Lys Ile Gly Asn 180 185 190 Leu Gln Pro Val Leu Gln Gly AlaMet Lys Leu 195 200 51 609 DNA Rat 51 atggaaatta tttcatcaaa gcgattcattttattgactt tagcaacttc aagcttctta 60 acttcaaaca ccctttgttc agatgaattaatgatgcccc attttcacag caaagaaggt 120 tatggaaaat attaccagct gagaggaatcccaaaagggg taaaggaaag aagtgtcact 180 tttcaagaac tcaaagattg gggggcaaagaaagatatta agatgagtcc agcccctgcc 240 aacaaagtgc cccactcagc agccaaccttcccctgaggt ttgggaggaa catagaagac 300 agaagaagcc ccagggcacg ggccaacatggaggcaggga ccatgagcca ttttcccagc 360 ctgccccaaa ggtttgggag aacaacagccagacgcatca ccaagacact ggctggtttg 420 ccccagaaat ccctgcactc cctggcctccagtgaattgc tctatgccat gacccgccag 480 catcaagaaa ttcagagtcc tggtcaagagcaacctagga aacgggtgtt cacggaaaca 540 gatgatgcag aaaggaaaca agaaaaaataggaaacctcc agccagtcct tcaaggggct 600 atgaagctg 609 52 27 DNA ArtificialSequence primer 52 ttctagattt tggacaaaat ggaaatt 27 53 27 DNA ArtificialSequence primer 53 cgtctttagg gacaggctcc agatttc 27 54 430 PRT Human 54Met Glu Gly Glu Pro Ser Gln Pro Pro Asn Ser Ser Trp Pro Leu Ser 1 5 1015 Gln Asn Gly Thr Asn Thr Glu Ala Thr Pro Ala Thr Asn Leu Thr Phe 20 2530 Ser Ser Tyr Tyr Gln His Thr Ser Pro Val Ala Ala Met Phe Ile Val 35 4045 Ala Tyr Ala Leu Ile Phe Leu Leu Cys Met Val Gly Asn Thr Leu Val 50 5560 Cys Phe Ile Val Leu Lys Asn Arg His Met His Thr Val Thr Asn Met 65 7075 80 Phe Ile Leu Asn Leu Ala Val Ser Asp Leu Leu Val Gly Ile Phe Cys 8590 95 Met Pro Thr Thr Leu Val Asp Asn Leu Ile Thr Gly Trp Pro Phe Asp100 105 110 Asn Ala Thr Cys Lys Met Ser Gly Leu Val Gln Gly Met Ser ValSer 115 120 125 Ala Ser Val Phe Thr Leu Val Ala Ile Ala Val Glu Arg PheArg Cys 130 135 140 Ile Val His Pro Phe Arg Glu Lys Leu Thr Leu Arg LysAla Leu Val 145 150 155 160 Thr Ile Ala Val Ile Trp Ala Leu Ala Leu LeuIle Met Cys Pro Ser 165 170 175 Ala Val Thr Leu Thr Val Thr Arg Glu GluHis His Phe Met Val Asp 180 185 190 Ala Arg Asn Arg Ser Tyr Pro Leu TyrSer Cys Trp Glu Ala Trp Pro 195 200 205 Glu Lys Gly Met Arg Arg Val TyrThr Thr Val Leu Phe Ser His Ile 210 215 220 Tyr Leu Ala Pro Leu Ala LeuIle Val Val Met Tyr Ala Arg Ile Ala 225 230 235 240 Arg Lys Leu Cys GlnAla Pro Gly Pro Ala Pro Gly Gly Glu Glu Ala 245 250 255 Ala Asp Pro ArgAla Ser Arg Arg Arg Ala Arg Val Val His Met Leu 260 265 270 Val Met ValAla Leu Phe Phe Thr Leu Ser Trp Leu Pro Leu Trp Ala 275 280 285 Leu LeuLeu Leu Ile Asp Tyr Gly Gln Leu Ser Ala Pro Gln Leu His 290 295 300 LeuVal Thr Val Tyr Ala Phe Pro Phe Ala His Trp Leu Ala Phe Phe 305 310 315320 Asn Ser Ser Ala Asn Pro Ile Ile Tyr Gly Tyr Phe Asn Glu Asn Phe 325330 335 Arg Arg Gly Phe Gln Ala Ala Phe Arg Ala Arg Leu Cys Pro Arg Pro340 345 350 Ser Gly Ser His Lys Glu Ala Tyr Ser Glu Arg Pro Gly Gly LeuLeu 355 360 365 His Arg Arg Val Phe Val Val Val Arg Pro Ser Asp Ser GlyLeu Pro 370 375 380 Ser Glu Ser Gly Pro Ser Ser Gly Ala Pro Arg Pro GlyArg Leu Pro 385 390 395 400 Leu Arg Asn Gly Arg Val Ala His His Gly LeuPro Arg Glu Gly Pro 405 410 415 Gly Cys Ser His Leu Pro Leu Thr Ile ProAla Trp Asp Ile 420 425 430 55 1290 DNA Human 55 atggaggggg agccctcccagcctcccaac agcagttggc ccctaagtca gaatgggact 60 aacactgagg ccaccccggctacaaacctc accttctcct cctactatca gcacacctcc 120 cctgtggcgg ccatgttcattgtggcctat gcgctcatct tcctgctctg catggtgggc 180 aacaccctgg tctgtttcatcgtgctcaag aaccggcaca tgcatactgt caccaacatg 240 ttcatcctca acctggctgtcagtgacctg ctggtgggca tcttctgcat gcccaccacc 300 cttgtggaca acctcatcactgggtggccc ttcgacaatg ccacatgcaa gatgagcggc 360 ttggtgcagg gcatgtctgtgtcggcttcc gttttcacac tggtggccat tgctgtggaa 420 aggttccgct gcatcgtgcaccctttccgc gagaagctga ccctgcggaa ggcgctcgtc 480 accatcgccg tcatctgggccctggcgctg ctcatcatgt gtccctcggc cgtcacgctg 540 accgtcaccc gtgaggagcaccacttcatg gtggacgccc gcaaccgctc ctaccctctc 600 tactcctgct gggaggcctggcccgagaag ggcatgcgca gggtctacac cactgtgctc 660 ttctcgcaca tctacctggcgccgctggcg ctcatcgtgg tcatgtacgc ccgcatcgcg 720 cgcaagctct gccaggccccgggcccggcc cccgggggcg aggaggctgc ggacccgcga 780 gcatcgcggc gcagagcgcgcgtggtgcac atgctggtca tggtggcgct gttcttcacg 840 ctgtcctggc tgccgctctgggcgctgctg ctgctcatcg actacgggca gctcagcgcg 900 ccgcagctgc acctggtcaccgtctacgcc ttccccttcg cgcactggct ggccttcttc 960 aacagcagcg ccaaccccatcatctacggc tacttcaacg agaacttccg ccgcggcttc 1020 caggccgcct tccgcgcccgcctctgcccg cgcccgtcgg ggagccacaa ggaggcctac 1080 tccgagcggc ccggcgggcttctgcacagg cgggtcttcg tggtggtgcg gcccagcgac 1140 tccgggctgc cctctgagtcgggccctagc agtggggccc ccaggcccgg ccgcctcccg 1200 ctgcggaatg ggcgggtggctcaccacggc ttgcccaggg aagggcctgg ctgctcccac 1260 ctgcccctca ccattccagcctgggatatc 1290 56 1290 DNA Human 56 atggaggggg agccctccca gcctcccaacagcagttggc ccctaagtca gaatgggact 60 aacactgagg ccaccccggc tacaaacctcaccttctcct cctactatca gcacacctcc 120 cctgtggcgg ccatgttcat tgtggcctatgcgctcatct tcctgctctg catggtgggc 180 aacaccctgg tctgtttcat cgtgctcaagaaccggcaca tgcatactgt caccaacatg 240 ttcatcctca acctggctgt cagtgacctgctggtgggca tcttctgcat gcccaccacc 300 cttgtggaca acctcatcac tgggtggcccttcgacaatg ccacatgcaa gatgagcggc 360 ttggtgcagg gcatgtctgt gtcggcttccgttttcacac tggtggccat tgctgtggaa 420 aggttccgct gcatcgtgca ccctttccgcgagaagctga ccctgcggaa ggcgctcgtc 480 accatcgccg tcatctgggc cctggcgctgctcatcatgt gtccctcggc cgtcacgctg 540 accgtcaccc gtgaggagca ccacttcatggtggacgccc gcaaccgctc ctacccgctc 600 tactcctgct gggaggcctg gcccgagaagggcatgcgca gggtctacac cactgtgctc 660 ttctcgcaca tctacctggc gccgctggcgctcatcgtgg tcatgtacgc ccgcatcgcg 720 cgcaagctct gccaggcccc gggcccggcccccgggggcg aggaggctgc ggacccgcga 780 gcatcgcggc gcagagcgcg cgtggtgcacatgctggtca tggtggcgct gttcttcacg 840 ctgtcctggc tgccgctctg ggcgctgctgctgctcatcg actacgggca gctcagcgcg 900 ccgcagctgc acctggtcac cgtctacgccttccccttcg cgcactggct ggccttcttc 960 aacagcagcg ccaaccccat catctacggctacttcaacg agaacttccg ccgcggcttc 1020 caggccgcct tccgcgcccg cctctgcccgcgcccgtcgg ggagccacaa ggaggcctac 1080 tccgagcggc ccggcgggct tctgcacaggcgggtcttcg tggtggtgcg gcccagcgac 1140 tccgggctgc cctctgagtc gggccctagcagtggggccc ccaggcccgg ccgcctcccg 1200 ctgcggaatg ggcgggtggc tcaccacggcttgcccaggg aagggcctgg ctgctcccac 1260 ctgcccctca ccattccagc ctgggatatc1290 57 31 DNA Artificial Sequence primer 57 gtcgacatgg agggggagccctcccagcct c 31 58 29 DNA Artificial Sequence primer 58 actagttcagatatcccagg ctggaatgg 29 59 58 DNA Artificial Sequence Primer 59tatgagcctg aactttgaag aactgaaaga ttggggtccg aaaaatgtga ttaaaatg 58 60 61DNA Artificial Sequence Primer 60 agcaccccgg cggtgaataa aatgccgcatagctttgcga atctgccgct gcgtttttgc 60 c 61 61 62 DNA Artificial SequencePrimer 61 ggtgctcatt ttaatcacat ttttcggacc ccaatctttc agttcttcaaagttcaggct 60 ca 62 62 59 DNA Artificial Sequence Primer 62 tcggggcaaaaacgcagcgg cagattcgca aagctatgcg gcattttatt caccgccgg 59 63 28 PRT Human63 Ala Thr Ala Asn Leu Pro Leu Arg Ser Gly Arg Asn Met Glu Val Ser 1 510 15 Leu Val Arg Arg Val Pro Asn Leu Pro Gln Arg Phe 20 25 64 84 DNAHuman 64 gcaacagcca acctgcctct gagatctgga agaaatatgg aggtgagcctcgtgagacgt 60 gttcctaacc tgccccaaag gttt 84 65 31 PRT Human 65 Ser AlaGly Ala Thr Ala Asn Leu Pro Leu Arg Ser Gly Arg Asn Met 1 5 10 15 GluVal Ser Leu Val Arg Arg Val Pro Asn Leu Pro Gln Arg Phe 20 25 30 66 93DNA Human 66 agtgctggag caacagccaa cctgcctctg agatctggaa gaaatatggaggtgagcctc 60 gtgagacgtg ttcctaacct gccccaaagg ttt 93 67 28 PRT Bovine67 Ala Met Ala His Leu Pro Leu Arg Leu Gly Lys Asn Arg Glu Asp Ser 1 510 15 Leu Ser Arg Trp Val Pro Asn Leu Pro Gln Arg Phe 20 25 68 84 DNABovine 68 gcgatggccc acctgcctct gagactcgga aaaaatagag aggacagcctctccagatgg 60 gtcccaaatc tgccccagag gttt 84 69 5 PRT Artificial SequencePeptide 69 Leu Pro Gln Arg Phe 1 5 70 6 PRT Artificial Sequence Peptide70 Asn Leu Pro Gln Arg Phe 1 5 71 7 PRT Artificial Sequence Peptide 71Pro Asn Leu Pro Gln Arg Phe 1 5 72 8 PRT Artificial Sequence Peptide 72Val Pro Asn Leu Pro Gln Arg Phe 1 5

1. An isolated RFamide-Related Peptide-3 (RFRP-3) peptide having anamino acid sequence selected from the group consisting of: (1) an aminoacid sequence which is from position 104 (Ala) to position 131 (Phe) ofSEQ ID NO: 1; (2) an amino acid sequence which is from position 101(Ser) to position 131 (Phe) of SEQ ID NO: 1; (3) an amino acid sequencewhich is from position 104 (Ala) to position 131 (Phe) of SEQ ID NO: 14;(4) an amino acid sequence which is from position 125 (Pro) to position131 (Phe) of SEQ ID NO: 1; (5) an amino acid sequence which is fromposition 126 (Asn) to position 131 (Phe) of SEQ ID NO: 1; or (6) anamino acid sequence which is from position 127 (Leu) to position 131(Phe) of SEQ ID NO: 1, or an amide or ester thereof, or a salt thereof.2. CANCELED.
 3. CANCELED.
 4. The peptide according to claim 1 whereinthe C-terminal carboxyl group is amidated, or a salt thereof.
 5. Anisolated polynucleotide comprising a polynucleotide encoding the peptideaccording to claim
 1. 6. CANCELED.
 7. The polynucleotide according toclaim 5, which is a DNA.
 8. The polynucleotide according to claim 5,having a nucleic acid sequence selected from the group which consistsof: (1) a nucleotide sequence which is from position 310 to position 393of SEQ ID NO: 2; (2) a nucleotide sequence which is from position 301 toposition 393 of SEQ ID NO: 2; (3) a nucleotide sequence which is fromposition 310 to position 393 of SEQ ID NO: 15; (4) a nucleotide sequencewhich is from position 373 to position 393 of SEQ ID NO: 2; (5) anucleotide sequence which is from position 376 to position 393 of SEQ IDNO: 2; or (6) a nucleotide sequence which is from position 379 toposition 393 of SEQ ID NO:
 2. 9. CANCELED.
 10. A recombinant vectorcomprising the polynucleotide according to claim
 5. 11. A transformanthost cell transformed with the recombinant vector according to claim 10.12. A method for producing the peptide according to claim 1 or an amideor ester thereof, or a salt thereof, comprising transforming a suitablehost cell with an expressible recombinant vector which comprises anucleic acid sequence encoding for said peptide, culturing thetransformant host cell under suitable conditions and for sufficient timeto express said peptide.
 13. A pharmaceutical composition forregulating, promoting or inhibiting prolactin secretion comprising aneffective amount of the peptide according to claim 1 or an amide orester thereof, or a salt thereof and a pharmaceutically acceptablecarrier excipient or diluent.
 14. A pharmaceutical composition forregulating, promoting or inhibiting prolactin secretion comprising aneffective amount of the polynucleotide according to claim 5 and apharmaceutically acceptable carrier, excipient or diluent.
 15. CANCELED.16. CANCELED.
 17. CANCELED.
 18. A pharmaceutical composition fortreating or preventing hypoovarianism, seminal vesicle hypoplasia,osteoporosis, menopausal syndrome, hypogalactia, hypothyroidism or renalinsufficiency comprising an effective amount of a peptide of claim 1, ora nucleic acid encoding for said peptide, and a pharmaceuticallyacceptable carrier, excipient or diluent.
 19. A pharmaceuticalcomposition for treating or preventing hyperprolactinemia, pituitaryadenoma, diencephalic tumor, menstrual disorder, stresses, autoimmunediseases, prolactinoma, sterility, impotence, amenorrhea, galactorrhea,acromegaly, Chiari-Frommel syndrome, Argonz-del Castillo syndrome,Forbes-Albright syndrome, breast cancer lymphoma, Sheehan's syndrome orspermatogenic disorder comprising an effective amount of a peptide ofclaim 1, or a nucleic acid encoding for said peptide, and apharmaceutically acceptable carrier, excipient or diluent.
 20. Apharmaceutical composition for promoting galactopoiesis in a mammalcomprising an effective amount of a peptide of claim 1, or a nucleicacid encoding for said peptide, and a pharmaceutically acceptablecarrier, excipient or diluent.
 21. A pharmaceutical composition fortesting the function of prolactin secretion in a mammal comprising aneffective amount of a peptide of claim 1, or a nucleic acid encoding forsaid peptide, and a pharmaceutically acceptable carrier, excipient ordiluent.
 22. An isolated antibody which specifically binds to a RFRP-3peptide according to claim 1 or an amide or ester thereof, or a saltthereof.
 23. A medicine comprising the antibody according to claim 22.24. The medicine according to claim 23, which is a prophylactic and/ortherapeutic agent for hyperprolactinemia, pituitary adenoma,diencephalic tumor, menstrual disorder, stresses, autoimmune diseases,prolactinoma, sterility, impotence, amenorrhea, galactorrhea,acromegaly, Chiari-Frommel syndrome, Argonz-del Castillo syndrome,Forbes-Albright syndrome, breast cancer lymphoma or Sheehan's syndromeor spermatogenic disorder.
 25. A diagnostic agent comprising theantibody according to claim
 22. 26. The diagnostic agent according toclaim 25, which is a diagnostic agent for hypoovarianism, seminalvesicle hypoplasia, osteoporosis, menopausal syndrome, hypogalactia,hypothyroidism or renal insufficiency, hyperprolactinemia, pituitaryadenoma, diencephalic tumor, menstrual disorder, stresses, autoimmunediseases, prolactinoma, sterility, impotence, amenorrhea, galactorrhea,acromegaly, Chiari-Frommel syndrome, Argonz-del Castillo syndrome,Forbes-Albright syndrome, breast cancer lymphoma or Sheehan's syndromeor spermatogenic disorder.
 27. A diagnostic agent comprising an isolatedRFRP-3 encoding polynucleotide according to claim
 5. 28. The diagnosticagent according to claim 27, which is a diagnostic agent forhypoovarianism, seminal vesicle hypoplasia, osteoporosis, menopausalsyndrome, hypogalactia, hypothyroidism or renal insufficiency,hyperprolactinemia, pituitary adenoma, diencephalic tumor, menstrualdisorder, stresses, autoimmune diseases, prolactinoma, sterility,impotence, amenorrhea, galactorrhea, acromegaly, Chiari-Frommelsyndrome, Argonz-del Castillo syndrome, Forbes-Albright syndrome, breastcancer, lymphoma or Sheehan's syndrome or spermatogenic disorder.
 29. Anisolated antisense DNA which comprises a nucleotide sequence, or a partthereof, complementary or substantially complementary to a DNA encodingfor a RFRP-3 peptide according to claim 1, and has an effect capable ofinhibiting the expression of said DNA.
 30. A medicine comprising theantisense DNA according to claim
 29. 31. The medicine according to claim30, which is a prophylactic and/or therapeutic agent forhyperprolactinemia, pituitary adenoma, diencephalic tumor, menstrualdisorder, stresses, autoimmune diseases, prolactinoma, sterility,impotence, amenorrhea, galactorrhea, acromegaly, Chiari-Frommelsyndrome, Argonz-del Castillo syndrome, Forbes-Albright syndrome, breastcancer lymphoma or Sheehan's syndrome or spermatogenic disorder.
 32. Amethod of screening for a compound or a salt thereof that promotes orinhibits the activity of a RFRP-3 peptide according to claim 1 or anamide or ester thereof, or a salt thereof, comprising using the peptideaccording to claim 1 or an amide or ester thereof, or a salt thereof ina screening assay.
 33. The screening method according to claim 32,further comprising using a protein comprising an amino acid sequenceidentical or substantially identical to the amino acid sequencerepresented by SEQ ID NO: 37 or a salt of said protein, or a partialpeptide of said protein, or an amide or ester of said partial peptide,or a salt of said partial peptide.
 34. The screening method according toclaim 32, further comprising using a protein consisting of the aminoacid sequence represented by SEQ ID NO: 37 or SEQ ID NO: 54 or a salt ofsaid protein, or a partial peptide of said protein, or an amide or esterof said partial peptide, or a salt of said partial peptide.
 35. A kitfor screening for a compound or a salt thereof that promotes or inhibitsthe activity of the peptide according to claim 1 an amide or esterthereof, or a salt thereof, comprising the peptide according to claim 1or an amide or ester thereof, or a salt thereof.
 36. A compound or asalt thereof that promotes or inhibits the activity of the peptideaccording to claim 1 or an amide or ester thereof, or a salt thereof,wherein said compound or salt thereof is obtainable by the screeningmethod according to claim
 32. 37. A medicine comprising a compound or asalt thereof that promotes or inhibits the activity of the peptideaccording to claim 1 or an amide or ester thereof, or a salt thereof.38. An agent for promoting prolactin secretion comprising a compound ora salt thereof that promotes the activity of the peptide according toclaim 1 or an amide or ester thereof, or a salt thereof.
 39. Aprophylactic and/or therapeutic agent for hypoovarianism, seminalvesicle hypoplasia, osteoporosis, menopausal syndrome, hypogalactia,hypothyroidism or renal insufficiency, comprising a compound or a saltthereof that promotes the activity of the peptide according to claim 1or an amide or ester thereof, or a salt thereof.
 40. An agent forpromoting galactopoiesis in a mammal, comprising a compound or a saltthereof that promotes the activity of the peptide according to claim 1or an amide or ester thereof, or a salt thereof.
 41. An inhibitor forprolactin secretion comprising a compound or a salt thereof thatinhibits the activity of the peptide according to claim 1 or an amide orester thereof, or a salt thereof.
 42. A prophylactic and/or therapeuticagent for hyperprolactinemia, pituitary adenoma, diencephalic tumor,menstrual disorder, stresses, autoimmune diseases, prolactinoma,sterility, impotence, amenorrhea, galactorrhea, acromegaly,Chiari-Frommel syndrome, Argonz-del Castillo syndrome, Forbes-Albrightsyndrome, breast cancer lymphoma, Sheehan's syndrome or spermatogenicdisorder comprising a compound or a salt thereof that modulates theactivity of the peptide according to claim 1 or an amide or esterthereof, or a salt thereof.
 43. A method of promoting prolactinsecretion, comprising administering to a mammal an effective amount of(i) the peptide according to claim 1 or an amide or ester thereof, or asalt thereof, (ii) the polynucleotide according to claim 5 or 6, or(iii) a compound or a salt thereof that promotes the activity of thepeptide according to claim 1 or an amide or ester thereof, or a saltthereof.
 44. A method of preventing and/or treating hypoovarianism,seminal vesicle hypoplasia, osteoporosis, menopausal syndrome,hypogalactia, hypothyroidism or renal insufficiency, comprisingadministering to a mammal an effective amount of (i) the peptideaccording to claim 1 or an amide or ester thereof, or a salt thereof,(ii) the polynucleotide according to claim 5 or 6, or (iii) a compoundor a salt thereof that promotes the activity of the peptide according toclaim 1 or F or an amide or ester thereof, or a salt thereof.
 45. Amethod of inhibiting prolactin secretion, comprising administering to amammal an effective amount of (i) the antibody according to claim 22,(ii) the antisense DNA according to claim 29, or (iii) a compound or asalt thereof that inhibits the activity of the peptide according toclaim 1 or an amide or ester thereof, or a salt thereof.
 46. A method ofpreventing and/or treating hyperprolactinemia, pituitary adenoma,diencephalic tumor, menstrual disorder, stresses, autoimmune diseases,prolactinoma, sterility, impotence, amenorrhea, galactorrhea,acromegaly, Chiari-Frommel syndrome, Argonz-del Castillo syndrome,Forbes-Albright syndrome, breast cancer lymphoma, Sheehan's syndrome orspermatogenic disorder, comprising administering to a mammal aneffective amount of (i) an antibody, (ii) an antisense DNA, or (iii) acompound or a salt thereof, that inhibits the activity of the peptideaccording to claim 1 or an amide or ester thereof, or a salt thereof.47. Use of (i) the peptide according to claim 1 or an amide or esterthereof, or a salt thereof, (ii) the polynucleotide that encodes for apolypeptide of claim 1, or (iii) a compound or a salt thereof thatpromotes the activity of the peptide according to claim 1 or an amide orester thereof, or a salt thereof, for manufacturing a prolactinsecretion-promoting agent.
 48. Use of (i) the peptide according to claim1 or an amide or ester thereof, or a salt thereof, (ii) apolynucleotide, or (iii) a compound or a salt thereof that promotes theactivity of the peptide according to claim 1 or 2 or an amide or esterthereof, or a salt thereof, for manufacturing a prophylactic and/ortherapeutic agent for hypoovarianism, seminal vesicle hypoplasia,osteoporosis, menopausal syndrome, hypogalactia, hypothyroidism or renalinsufficiency.
 49. Use of (i) an antibody, (ii) an antisense DNA, or(iii) a compound or a salt thereof, that inhibits the activity of thepeptide according to claim 1 or an amide or ester thereof, or a saltthereof, for manufacturing a prolactin secretion inhibitor.
 50. Use of(i) an antibody, (ii) an antisense DNA, or (iii) a compound or a saltthereof, that inhibits the activity of the peptide according to claim 1or an amide or ester thereof, or a salt thereof, for manufacturing aprophylactic and/or therapeutic agent for hyperprolactinemia, pituitaryadenoma, diencephalic tumor, menstrual disorder, stresses, autoimmunediseases, prolactinoma, sterility, impotence, amenorrhea, galactorrhea,acromegaly, Chiari-Frommel syndrome, Argonz-del Castillo syndrome,Forbes-Albright syndrome, breast cancer lymphoma, Sheehan's syndrome orspermatogenic disorder.